THE  PREPARATION.  PROPERTIES  AND  METABOLIC 
BEHAVIOR  OF  DEAMINIZED  PROTEINS 


MAX  SHAW  DUNN 

A.  B.  Simpson  College,  1916 
M.  S.  University  of  Illinois,  1918 


THESIS 

Submitted  in  Partial  Fulfillment  of  the  Requirements  for  the 

Degree  of 

DOCTOR  OF  PHILOSOPHY 

IN  CHEMISTRY 
IN 

THE  GRADUATE  SCHOOL 
OF  THE 

UNIVERSITY  OF  ILLINOIS 
1921 

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1. 


INTRODUCTION. 

One  of  the  important  prohlems  involved  in  the  proof  of 
the  structure  of  the  protein  molecule  is  that  of  the  nature 
of  its  basicity.  It  is  believed  that  native  proteins  are 
basic  because  of  amino  groups  which,  instead  of  being  in 
peptide  linkage  with  carboxyl  groups,  exist  free  in  the 
molecule.  In  endeavoring  to  ascertain  which  of  the  various 
amino  acids  contain  the  amino  groups  responsible  for  the 
basicity  of  native  proteins,  Skraup  (l)  found  that  lysine 
and  tyrosine  were  lacking  in  proteins  which  had  been  deamin- 
ized with  nitrous  acid  while  arginine  and  histidine  were 
present  in  diminished  quantities.  Therefore  the  assumption 
that  lysine  and  tyrosine  and  possibly  arginine  and  histidine 
contain  amino  groups  free  in  the  protein  molecule  seemed 
tenable.  Kossel  and  Cameron  (2)  have  shown  that  arginine  does 
have  an  amino  groun  free  in  the  protein  molecule  but  that 
this  group  is  contained  in  the  guanidine  nucleus  which  does  l 
not  react  with  nitrous  acid.  Van  Slyke  (3)  and  others  (4) 
have  found  that  the  nitrogen  liberated  from  native  proteins  i 
by  the  action  of  nitrous  acid  is  approximately  one-half  of 
the  lysine  nitrogen  present.  Because  of  these  observations. 

Van  Slyke  has  put  forth  the  theory  that  the  free  amino  nitro- 
gen of  native  proteins  is  due  almost  entirely  to  one  of  the 
amino  groups  of  lysine.  Since  it  requires  thirty  minutes  for 
the  deaminization  of  native  proteins  and  for  the  cleavage  of 


:fb. 


Digitized  by  the  Internet  Archive 
in  2016 


https://archive.org/details/preparationpropeOOdunn 


the  epsilon  amino  groun  of  lysine  hy  this  reagent,  the  free 
amino  groups  of  native  proteins  are  commonly  thought  to  he 
due,  almost  all  if  not  entirely,  to  the  terminal  amino  group 
of  lysine.  However,  it  was  the  view  of  Kossol  (5)  that  the 
existence  of  a quantitative  relation  hetween  the  free  amino 
nitrogen  and  the  lysine  content  of  a protein  was  improbable . 
Evidence  in  supnort  of  this  view  has  recently  been  obtained 
by  Edlbacher  (6)  and  by  Felix  (7).  The  former  investigator 
in  studying  the  action  of  methylating  agents  upon  native  pro- 
teins found  that  the  lysine  free  proteins,  clupeine  and  sal- 
mlne,  methylated  as  easily  as  other  lysine  containing  proteins, 
while  formaldehyde  reacted  only  with  the  proteins  of  the  latter 
character.  These  observations  led  to  the  conclusion  that 
there  are  free  amino  groups  in  the  protein  molecule  other 
than  that  of  lysine.  Herzig  (8)  has  recently  reported  that 
deaminized  gelatin  is  methylated  as  easily  as  gelatin  itself. 
These  observations  would  seem  to  establish  the  existence  of 
groups  in  the  protein  molecule,  other  than  the  free  amino 
group  of  lysine,  which  are  capable  of  methylation.  However, 
it  does  not  follow  of  necessity  that  the  groups  which  have 
been  methylated  are  free  amino  in  character.  Additional  ob- 
jections to  the  theory  proposed  by  Van  Slyke  have  been  raised 
by  Felix,  who  believes  that  the  lysine  determination  by  the 
Van  Slyke  partition  method  gives  high  values  for  this  amino 
acid  ov/ing  to  the  precipi tation  by  phospho tungstic  acid  of 
substances,  such  as  phenyl  alanine  or  proline,  in  addition  to 
the  amino  acids  of  the  hexone  bases.  If,  on  the  other  hand, 
the  figures  given  for  lysine  by  the  Kossel  method  of  direct 


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3. 


isolation  are  accented,  one  half  of  the  lysine  nitrogen  of  the 
proteins  investigated  will  not  evon  approximate  the  free 
amino  nitrogen  as  determined  by  the  nitrous  acid  method.  This 
author  wishes  to  note  further  that  the  values  for  the  free 
amino  nitrogen  of  certain  proteins,  particularly  gelatin,  as 
determined  by  the  nitrous  acid  method  and  by  formol  titration 
are  not  in  agreement.  Furthermore,  it  was  found  by  these  in- 
vestigators in  the  case  of  histones,  sturin,  gelatin  and  gly- 
cinin  that  the  free  amino  nitrogen  as  determined  by  the 
nitrous  acid  method  so  exceeded  one  half  of  the  lysine  nitro- 
gen of  these  nroteins  that  the  existence  of  free  amino  groups 
other  than  that  due  to  lysine  seemed  probable. 

The  problem  of  the  basicity  of  the  protein  molecule  has 
been  attac-^ed  in  another  way  by  studying  the  acid-combining 
capacity  of  proteins.  Robertson  (9)  believes  that  the  ex- 
periments of  Blasel  and  Matula  and  those  of  Pauli  and  Hirsch- 
feld  are  ” direct  proof  that  the  terminal  groups  are  not 

responsible  for  any  apDreciable  oortion  of  the  acid  combining- 

capacity  of  proteins.  These  investigators  found  that 

the  combining-caoaci ty  of  deaminized  gelatin  for  acids  is  but 
slightly  inferior  to  that  of  ordinary  gelatin,  indicating, 
beyond  any  question,  that  the  combining-capaci ty  of  gelatin 
for  acids,  is  in  very  large  proportion,  attributable  to  ele- 
ments of  the  molecule  other  than  the  free  -NH  groups.  The 
inference  is  unavoidable  that  the  elements  of  the  molecule 
which  actually  participate  in  the  union  with  acids  are,  in 
very  large  pronortion,  the  -COHN-  groups  within  the  body  of 
the  protein  molecule.”  Bracewell  (10)  is  of  the  opinion 


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4. 


that  ” the  amount  of  acid  'neutralized’  "by  a considerable 
number  of  proteins  is  determined  by  the  free  amino  groups  in 
the  protein  molecule."  By  taking  into  account  the  possibility 
of  a free  amino  group  in  arginine,  this  investigator  believes 
that  "the  amount  of  acid  absorbed  by  ordinary  proteins  is 
determined  mainly  by  their  content  of  lysine  and  arginine." 

In  the  oresent  investigation  it  was  considered  to  be  of 
interest  and  iraoortance  to  make  a careful  study  of  a typical 
deaminized  protein,  i.e.  deaminized  casein,  and  to  determine 
to  what  extent  the  amounts  of  various  amino  acids,  the  dis- 
tribution of  nitrogen  in  the  molecule,  the  metabolic  behavior 
and  other  properties  had  been  altered  by  the  processes  of 
deaminization. 


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5. 


EXPERIMENTAL . 

A.  The  PreDaration  of  Casein  and  Deaminized  Casein . 

Although  the  original  observations  on  the  preparation  of 
casein  from  milk  were  made  by  Hoppe  (11)  in  1359,  it  remained 
for  Hammarsten  (12),  in  1877,  to  accurately  describe  the  pre- 
paration and  nurification  of  this  protein.  Since  that  time 
various  modifications  (13)  of  Hammarsten ' s method  have  been 
proDOsed. 

The  procedure  used  in  the  present  study  is  an  adaptation 
of  the  more  modern  methods  for  casein  preparation.  This 
method  is  given  in  detail.  Two  gallons  of  skim  milk  are  added 
to  10  liters  of  distilled  v/ater  contained  in  a carboy  of 
about  50  liters  capacity.  Dilute  acetic  acid,  made  by  adding 
6 c.c.  of  glacial  acetic  acid  to  a liter  of  distilled  water, 
is  very  slowly  added  to  this  water-milk  mixture,  which  is 
mixed  uniformly  by  stirring  with  a current  of  air.  After  the 
addition  of  5 to  6 liters  of  the  acetic  acid  solution,  the 
casein  flocks  out  completely  and  settles  ranidly  to  the  bottom 

I 

of  the  carboy.  The  yellowish,  supernatant  liquid  is  siphoned 
off  and  the  precipitated  casein  washed  with  10  or  more  liters 
of  distilled  water;  the  supernatant  liquid  is  again  siphoned 
off  and  the  washing  process  repeated.  The  casein  remaining 
from  the  final  washing  is  dissolved  by  the  addition  of  a 
solution  of  dilute  ammonium  hydroxide,  containing  6 c.c.  of 
concentrated  ammonium  hydroxide  to  a liter  of  distilled  water. 
About  7 liters  of  alkali  will  completely  dissolve  the  casein 


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to  give  an  onalescent  solution,  which  is  then  filtered  through 
a thick  layer  of  absorbent  cotton.  The  whole  process  of  pre- 
cipitating with  acid  and  dissolving  in  alkali  is  repeated  3 
times.  After  the  last  precipi tation,  the  casein  is  washed 
tvace  with  distilled  v/ater,  the  sunernatant  liquid  decanted 
and  the  purified  product  filtered  on  a Buchner  funnel  using 
hardened  filter  naper  and  suction.  The  compact  cake,  which 
is  formed,  is  triturated  in  a mortar  with  95%  alcohol  and  the 
trituration  with  alcohol  is  repeated  3 times.  After  tritur- 
ating 3 times  with  dry  ether  to  completely  dehydrate  the 
casein  cake  theproduct  is  dried  for  3 hours  in  air  and  finally 
for  30  minutes  in  the  oven  at  80°  C.  The  resulting  white 
product  may  be  powdered  to  a dust  in  a ball  mill  or  by  passing 
it  through  an  80  mesh  sieve. 

Nitrous  acid  was  first  used  as  a deaminizing  agent  for 
proteins  in  1885  by  Loew  (14).  This  investigator  found  that 
one-third  of  the  nitrogen  of  peptones  was  liberated  by  the 
action  of  nitrous  acid.  In  1896  Paal  (15)  used  silver  nitrite 
and  hydrochloric  acid  to  deaminate  gelatin  peptones,  while  in 
the  same  year  Schiff  (16)  obtained  a straw  yellow  compound  by 
the  interaction  of  nitrous  acid  and  egg  albumin.  Two  years 
later  SchrStter  (17)  observed  the  formation  of  a similar  sub- 
stance from  peptones.  In  1908,  Treves  and  Salomons  (18)  al- 
lowed nitrous  acid  to  react  with  egg  albumin  and  obtained  a 
yellow  product  which  they  believed  was  diazo  albumin.  De- 
aminized gelatin  was  obtained  by  Blasel  and  iiatula  (19)  in 


1914. 


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f fi*''-L-fe«  /’0r\  ..r.  <l^C  . •>■♦  ihl  •'? 


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7. 


SkrauD  (20)and  his  pupils  have  prepared  and  studied  the 
deaminized  products  of  the  following  proteins:  casein  (1); 

gelatin  (21);  albumin  (22);  globulin  (23);  and  edestin  (24). 
Deaminized  albumin,  gelatin,  and  casein  (25)  and  deaminized 
gliadin  and  vitellin  (26)  have  been  prepared  and  investigated 
by  Levites. 

Levites  and  Skraup  have  been  largely  responsible  for 
perfecting  the  methods  used  in  preparing  deaminized  protein 
products.  In  the  first  (25)  method  of  Levites,  a paste  made 
from  the  nrotein  and  sodium  nitrite  was  warmed  on  the  water 
bath  with  dilute  acetic  acid  and  the  resulting  olive  green 
product  was  dried  in  vacuo.  In  his  second  (25)  procedure, 
Levites  produced  an  emulsion  of  the  protein  by  agitating  it 
vigorously  in  a shaking  machine  with  10^  acetic  acid.  This 
emulsion  was  warmed  gently  on  the  water  bath  with  a 10^  solu- 
tion of  sodium  nitrite  and  the  resulting  yellow  product  fil- 
tered, washed  with  alcohol  and  ether  and  dried  in  vacuo.  In 
the  method  used  by  Skraup  (1)  an  acid  solution  of  the  protein, 
prepared  by  adding  glacial  acetic  acid  to  a uniform  suspension 
of  the  protein  in  water,  was  warmed  gently  with  sodium  nitrite 
on  the  water  bath  and  the  yellow  product  drained  off  on  linen, 
desiccated  and  dried  in  air. 

In  the  present  series  of  experiments  deaminized  casein 
was  prepared  according  to  the  methods  outlined  by  Levites  and 
by  Skraup.  It  was  difficult  to  obtain  a product  of  uniform 
color  and  appearance  while  the  yield  of  70  percent  reported 
by  Skraup  was  rarely  exceeded.  There  are  several  objections 
to  the  use  of  a shaking  machine  as  employed  by  Levites.  It 


. V* 


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8. 


requires  several  hours  to  emulsify  small  amounts  of  protein 
and  the  emulsified  product  invariably  contains  gelatinous 
lumps  which  must  he  ground  uo  in  a mortar  to  avoid  contamin- 
ation of  the  deaminized  product  with  unchanged  protein.  All 
of  the  methods  used  for  the  nraparation  of  deaminized  pro- 
teins employ  heat  up  to  40°  C.  to  effect  complete  deaminiza- 
tion of  the  protein.  It  is  possible  that  this  apolication  of 
heat  may  cause  a slight  hydrolysis  of  the  protein. 

It  is  believed  that  the  following  procedure  overcomes 
the  objections  cited  above.  100  grams  of  casein  were  added  to 
2 liters  of  distilled  water  contained  in  a 5-liter  Pyrex 
flask.  After  stirring  vigorously  for  30  minutes  with  an 
electric  stirrer  a uniform  suspension  of  the  protein  resulted. 
To  this  suspension  140  c.c.  of  glacial  acetic  acid  were  added 
dropwise,  with  continued  stirring,  during  the  course  of  1.5 
hours.  At  the  expiration  of  20  minutes  a good  emulsion  was 
formed  while  at  the  end  of  the  period  a solution  was  effected. 
To  this  solution,  500  c.c.  of  a solution  of  sodium  nitrite 
containing  80  grams  to  a liter  was  added  dropwise,  with  con- 
tinued stirring,  during  the  space  of  1.5  hours.  After  150 
c.c.  of  this  solution  had  been  added  a deep  yellow  precipitate 
rose  to  the  top  of  the  liquid  as  a yellow  layer  which,  after 
standing  for  18  hours,  was  filtered  on  a Buchner  using  suction 
and  a hardened  filter  paper.  After  triturating  this  substance 
15  times  with  hot  water  to  the  disappearance  of  an  acid  re- 
action to  litmus,  it  was  granular  and  light  yellow  in  color 
while  the  aqueous  filtrate  was  similarly  colored.  The  yellow 


'/ 


i 

h 


precipitate  obtained  by  triturating  four  times  with  95% 
alcohol  was  thoroughly  desiccated  by  triturating  three  times 
with  dry  ether,  drying  in  air  for  30  minutes  and  in  the  oven 
at  80°  C.  for  an  equal  length  of  time.  Although  the  alcoholic 
filtrate  was  highly  colored  the  precipitate  appeared  to  have 
lost  but  little  of  its  yellow  color  in  the  washing  process. 

The  deaminized  product  was  of  a uniform  color  and 
appearance  and  it  was  possible  to  secure  practically  a com- 
plete transformation  into  the  deaminized  form.  From  three 
100-gram  samples  of  the  original  casein  yields  of  90,  95  and 
97.5  grams  of  the  oven  dried  product  were  obtained.  These 
deaminized  products  designated  subsequently  as  deaminized 
caseins  A-64,  A-66  and  A-68  were  very  fine  and  pov/dery  when 
passed  through  an  80-mesh  sieve.  They  were  colored  light 
yellow  when  first  prepared  but  surfaces  exposed  to  the  light 
for  a time  became  light  brown. 

With  the  second  method  of  Levites,  17  grams  of  deaminized 
casein  (A  - 18)  were  prepared  from  15  grams  of  casein.  How- 
ever, instead  of  permitting  a complete  deaminization  to 
occur,  the  white  precipitate  which  was  formed  upon  the  addi-  ! 
tion  of  the  sodium  nitrite  solution  was  filtered  on  a Buchner  ! 

as  soon  as  possible.  This  precipitate  which  was  triturated 
14  times  with  hot  water,  3 times  with  95^  alcohol,  and  twice 
with  dry  ether,  was  only  faintly  colored  yellow. 


10. 


B.  The  ProT)ertles  of  Deamlnl zed  Proteins . 

1.  Color.  Without  exceotion,  the  deaminized  protein 
nroducts  cited  in  the  literature  are  yellow  in  color.  Treves 
and  Salomons  (18)  believed  that  these  substances  were  diazo 
derivatives  since  they  responded  to  the  reactions  given  by 
diazo  compounds.  It  is  known,  however,  that  primary  aliphatic 
amines  do  not  react  with  nitrous  acid  under  ordinary  condi- 
tions to  give  stable  diazo  derivatives.  On  the  other  hand, 
were  this  a reaction  with  the  amide  groups  which  are  nresent 
acids  would  be  produced  (27)  and  not  diazo  products.  There- 
fore, the  assum-ntion  of  these  authors  seems  untenable. 

It  is  possible  that  deaminized  proteins  are  colored  be- 
cause of  the  formation  of  nitroso  compounds.  There  are  numer- 
ous possibilities  for  nitrosation  in  the  protein  molecule. 
Histidine,  tryptophane  and  proline  each  have  one  imino  nitro- 
gen, while  there  are  two  such  nitrogens  in  the  guanidine  group 
of  arginine.  Phenyl  alanine  would  admit  of  nitrosation  in 
the  para  position  of  its  benzene  nucleus,  while  a nitroso 
group  might  enter  tyrosine  in  the  position  ortho  to  the  hy- 
droxy group  in  the  benzene  ring.  It  is  unlikely  that  a nitro- 
sation of  the  imide  nitrogen  making  un  the  peptide  linkage 
has  taken  nlace  because  this  imide  is  probably  of  insufficient 
basicity,  due  to  the  neutralizing  action  of  the  adjacent  car- 
bonyl group,  to  react  with  nitrous  acid.  However  it  has  re- 
cently been  sho\vn  (23)  that  the  nitroso  derivative  of  methyl 
phthalimidine  is  easily  formed  by  treatment  with  nitrous  acid 
in  water  solution.  Since  the  carbonyl  imide  linkage  present 
in  methyl  phthalimidine  is  the  same  as  that  found  in  peptides 


11 


a possible  nitrosation  of  the  peptide  linkage  is  suggested. 
However,  this  cannot  have  taken  place  to  any  narked  extent 
because  deaminized  nroteins  have  been  shown  to  contain  less 
nitrogen  than  the  original  substances. 

Preliminary  to  carrying  out  some  qualitative  exneriments 
upon  several  nure  amino  acids,  it  was  found  that  the  red  solu- 
tion formed  by  the  action  of  nitrous  acid  upon  phenol  responds 
to  Liebermann’s  nitroso  reaction  by  giving  a green  solution 
with  concentrated  sulnhuric  acid.  Tyrosine  was  found  to  give 
a similar  reaction  after  warming  the  solution  gently  while 
indole  reacts  in  much  the  same  way  although  the  resulting 
color  is  of  lesser  intensity.  Histidine  and  phenyl  alanine 
give  no  red  color  with  sodium  nitrite  and  glacial  acetic  acid 
but  upon  the  further  addition  of  concentrated  sulphuric  acid 
a green  color  is  nroduced.  It  would  appear  from  tnese  tests 
that  the  yellow  to  brown  color  of  deaminized  proteins  may  be 
due  in  part  to  nitroso  derivatives  of  tyrosine  and  possibly 
tryptophane.  Prom  the  fact  that  gelatine,  a tyrosine  free 
protein,  is  renorted  to  give  yellow  deaminized  derivatives, 
it  would  seem  that  this  color  cannot  be  due  alone  to  the  | 

formation  of  nitroso  derivatives  of  tyrosine.  However,  it  is 
well  known  that  samples  of  gelatin,  considered  to  be  tyrosine 
free,  still  respond  to  Millon's  test  and  Dakin  (29)  has  re- 
cently found  traces  of  tyrosine  in  all  specimens  of  gelatin 
examined. 

2.  Solubility.  Deaminized  proteins  are  reported  to  be 
insoluble  in  water  and  insoluble  or  only  slightly  soluble  in 
alkalies.  Skraup  noted  the  formation  of  a jelly-like  substance 

" — " -«i 


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12. 


when  deaminized  proteins  were  brought  into  contact  with  strong 
alkali.  It  was  found  in  the  present  investigation  that  de- 
aminized casein  dissolved  in  .5^  sodium  hydroxide  after 
standing  for  two  days  with  the  formation  of  a red  solution 
and  a small  amount  of  undissolved  residuum.  With  1.5!^ 
sodium  hydroxide  solution,  a red  solution  was  formed  in  24 
hours,  while  with  concentrated  alkali  an  orange  to  brown 
jelly  was  formed  in  a fev;  minutes. 

3.  Color  Reactions.  Deaminized  casein  prepared  ac- 
cording to  the  method  described  above,  gave  positive  tests 
with  the  Hopkins-Cole , biuret  and  Millon's  reagents.  Millon's 
test  was  unquestionably  positive,  although  the  color  was  less 
intense  than  with  casein,  but  the  biuret  reaction  with  de- 
aminized casein  was  not  characteristic  ranging  from  a pink  to 
a reddish  purnle  color.  Levites  is  the  only  investigator  to 
report  a positive  Millon's  reaction  with  deaminized  proteins, 
while  the  biuret  reaction  was  found  to  be  positive  by  Levites 
and  by  Treves  and  Saloraone.  If  it  be  true  that  deaminized 
proteins  give  a positive  biuret  reaction,  this  would  indicate 
that  the  amide  grouping  which  is  responsible  for  the  color 

is  not  attacked  or  at  least  is  only  partially  destroyed  by 
the  action  of  nitrous  acid.  This  grouping,  according  to 
Schiff , is  two  CONH  groups  in  union. 

4.  Composition.  The  elementary  composition  of  native 
proteins  appears  to  be  but  little  altered  in  the  deaminiza- 
tion process.  Skraup  (20)  found  a slight  diminution  in  the 
phosphorous  content  of  deaminized  casein  and  a constant  in- 
crease, with  one  excention,  in  the  oxygen  content  of  all  of 


Pwf  ' ■ . • V 

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13. 


li 


the  deaminized  -oroteins  studied  hut  neither  observation  was 
considered  to  be  of  particular  signif icance . It  is  striking, 
however,  that  the  nitrogen  content  of  deaminized  proteins  is 
lower  than  that  of  the  original  substance.  Schiff  (16)  re- 
ports a reduction  of  1 percent  in  the  values  for  nitrogen 
while  the  figures  quoted  by  Skraup  (20) range  from  .51  to  1.24 
per  cent  lov/er  in  nitrogen  than  those  of  the  original  proteins. 
In  the  case  of  edestin  (24)  for  some  unaccountable  reason 
the  nitrogen  of  the  deaminized  product  was  found  to  be  higher 
than  that  of  the  original  protein. 

It  will  be  noted  from  Table  I that  the  nitrogen  content 
of  the  deaminized  casein  prepared  in  this  research  ranges  for 
the  various  sa’^nles  from  .22  to  .68  per  cent  lower  than  the 
figures  given  for  the  original  casein. 

TABLE  I. 

Nitrogen  calc,  on 


Samnle 

Ash 

an  ash  free  basis 

io 

Casein  1 

.38 

14.56 

Deaminized 

casein 

A-18 

.96 

13.91 

Deaminized 

casein 

A-64 

1.21 

14.34 

Deamini zed 

casein 

A-66 

.53 

13.88 

Deaminized 

. casein 

. A-63 

.49 

14.01 

5.  Free  Amino  Nitrogen.  The  first  apnlication  of  the 
reaction  between  nitrous  acid  and  amino  compounds  was  made  by 
Sachsse  and  Korraann  (30)  in  1874.  The  volume  of  nitrogen  lib- 
erated by  the  action  of  nitrous  acid  upon  plants  was  considered 


14. 


to  be  a measure  of  the  amide  grouos  present.  In  more  recent 
times  Brown  and  Willar  (31)  have  studied  this  reaction  further 
and  have  made  certain  improvements  unon  the  apparatus  used  by 
the  original  investigators.  It  remained,  however,  for  Van 
Slyke  (32)  in  1909  to  perfect  the  method  for  purifying  and 
measuring  the  nitrogen  evolved  in  this  reaction.  Because 
of  the  fact  that  amides  react  very  slowly  or  not  at  all  with 
nitrous  acid  under  the  conditions  employed,  it  is  believed 
that  the  nitrogen  liberated  from  proteins  and  from  protein 
derivatives  comes  entirely  from  the  primary  aliphatic  amines 
existing  free  in  the  molecule. 

The  determination  of  the  free  amino  nitrogen  of  native 
proteins  is  one  of  the  many  applications  (33)  which  have  been 
made  of  the  Van  Slyke  procedure.  In  the  present  study  of 
native  proteins  and  their  derivatives,  the  free  amino  nit- 
rogen of  casein,  deaminized  casein  and  gelatin  was  determined 
according  to  the  method  outlined  by  Van  Slyke  (34)  and  using 
the  micro  apparatus.  To  a suspension  of  3 grams  of  the  pro- 
tein in  distilled  water  was  added  a solution  containing  .375 
grams  of  sodium  carbonate.  The  proteins  went  completely  in 
solution  usually  within  an  hour.  This  protein  solution  was 
transferred  to  a 100  c.c.  volumetric  flask,  diluted  to  the 
mark  and  2 c.c.  taken  for  amino  nitrogen  analysis.  The 
casein  or  gelatin  solution  formed  in  this  manner  is  neutral 
to  litmus  and  Van  Slyke  (34)  has  shown  that  an  inappreciable 
hydrolysis  takes  place  even  after  standing  at  room  temperature 
for  48  hours.  In  the  deaminization  of  casein  a foam  inhibitor 
was  found  to  be  indispensable . For  this  purpose,  caprylic 


15. 


alcohol  was  found  to  he  more  effective  than  di  phenyl  ether, 
although  the  blank  resulting  from  the  former  substance  was, 
as  has  been  recorted  (35),  considerably  higher  than  that  given 
by  di  phenyl  ether.  Even  without  the  caprylic  alcohol,  the 
blank  from  the  sodium  nitrite  was  in  general  higher  than  that 
reported  by  Van  Slyke  for  good  samples  of  this  substance. 

Casein  was  found  to  nrecipitate  from  solution  immediately 
upon  contact  with  the  acid  solution  in  the  deaminizing  cham- 
ber and  to  gradually  change  from  a pure  white  to  yellow. 
Because  of  the  fact  that  casein  must  undergo  deamini zation 
while  in  the  solid  state  reliable  results  are  to  be  obtained 
only  by  maintaining  constant  conditions.  Uniform  results 
were  secured  by  shaking  the  deaminizing  chamber  for  30  minutes 
at  300  vibrations  ner  minute.  Gelatin  is  not  precioi tated  by 
the  acid  solution  of  the  deaminizing  chamber  and  does  not  im- 
part a color  to  the  liquid  during  the  process  of  deaminization . 

Van  Slyke  (3)  has  renorted  that  the  free  amino  nitrogen 
of  casein  comnrises  5.51  ner  cent  of  its  total  nitrogen  con- 
tent. Since  there  are  various  methods  which  are  used  for 
the  preparation  of  casein  from  cow's  milk,  it  was  considered 
of  importance  to  determine  the  free  amincjbi trogen  of  samples 
of  casein  prepared  in  a variety  of  ways  on  the  assumption 
that  the  reagents  emnloyed  in  the  purification  of  this  protein 
might  possibly  have  altered  its  free  amino  groups.  However, 
the  results  which  were  obtained  (see  Table  II)  were  not 
widely  divergent  and  it  would  seem  that  the  reagents  employed 
in  the  prenaration  of  these  samples  of  casein  were  without 
appreciable  influence  unon  the  number  of  free  amino  groups 


Vi-  irtif 


f*  ' I ‘ ^ \ *■  '.-'  ^'  , . S'y  ‘ ■ VV 

;.t'-;  '0iije£rOt»,y  d ^ 

I ' ■,  ' '' . -Jp. -\  ■ ,'T-'' ‘■■‘♦Jr  , 


8‘' 

i • * ' ’.'f:'/^Y%  *_‘rt--A>*r  ' ^ ^ .’1  I*  ffu  r W “'^Ur'JOsrV)  a.-  ^*rf.  ■ " _ . ' ..^^■•'t' /^.J  t , I 


'r  f 


16. 

of  this  protein.  It  should  be  noted,  however,  that  the 
average  figure  for  the  free  amino  nitrogen  content  of  ten 
samnles  of  casein  was  found  to  be  5.37  ner  cent  which  is  only 
slightly  higher  than  the  5.51  per  cent  reported  by  Van  Slyke . 

Casein  A-18,  which  was  only  slightly  yellow  in  color, 
was  prepared  by  treatment  with  nitrous  acid  but  was  removed 
from  the  influence  of  this  reagent  as  soon  as  nossible  after 
nrecipi tation . The  color  reactions  given  by  this  nroduct 
were  unquestionably  nositive  and  quite  comparable  to  the  re- 
actions given  by  casein  itself.  As  is  given  in  Table  II,  the 
free  amino  nitrogen  of  this  product  was  3.10  per  cent  of  the 
total  nitrogen,  while  that  of  completely  deaminized  casein 
A-64  was  0.0.  Since  the  value  for  casein  A-18  lies  midway 
between  that  given  by  unchanged  casein  on  one  hand  and  com- 
pletely deaminized  casein  on  the  other,  it  would  seem  that 
at  the  time  of  nrecipi tation  only  partial  deamini zation  had 
occurred. 

The  free  amino  nitrogen  content  of  two  samples  of  gelatin 
was  determined  and  found  to  be  5.29  and  5.41  per  cent  of  the 
total  nitrogen  figure.  These  values  are  in  agreement  with 
the  figure,  5.2  per  cent,  which  was  reported  by  Felix  (7) 
but  differ  from  the  nercent,  3.12,  found  by  Van  Slyke  (3). 

6.  Total  Amino  Nitrogen.  The  conditions  necessary  for 
the  complete  hydrolysis  of  proteins  have  been  determined. 
Comparable  results  have  been  secured  by  heating  in  an  auto- 
clave at  150  ° C.  for  1.5  hours  with  3.0  N hydrochloric  acid 
(37)  and  by  boiling  at  100*^  C.  for  24  to  43  hours  with  20^ 


1 


17. 


TABLE  II. 


Casein, 

Casein, 

Casein, 

Casein, 

Casein, 

If 

II 

ft 

If 

ft 


The  Free  Amino  Nitrogen  Content 
of  Casein,  Deaminized  Casein  and  Gelatin. 

Percent  of  total  nitrogen 


Samnles  as  free  amino  nitrogen 

Kahlhaum  5.61 

Kahlhaum  nach  Hammersten  5.99 

after  Van  Slyke  and  Bosworth  (13)  5.63 

after  Baker  and  Van  Slyke  (36)  5.52 

Oshorne  (#  ) No.  1 6.13 

” "2  5.84 

" ”3  6.24 

” ”4  6.22 

" "5  6.04 

" ”6  5.47 


'Deaminized*  casein,  A-18  3.10 
Deaminized  casein,  A-64  .00 
Gelatin,  renurified  Gold  Seal  5.29 
Gelatin,  Gold  Seal  5.41 


# Casein  samples  No.  1 to  No.  6 were  secured  through 
the  kindness  of  Prof.  T.  B.  Osborne  of  the  Connecticut 
Agriculture  Experiment  Station,  New  Haven,  Connecticut. 

In  every  case  unusual  procedures  were  used  in  the  prepara- 
tion of  these  samnles. 


-w. 


k: 


r 


•( 


V 


■V,., 


&, 


* ^ w 


'■r:.r  . 


( 


f 


i . 


V 


('f' 


y 

I ' • < ■ 

r • 

^ • . ' » 


► T 


-»x-m  , 


‘U,  ti.'Ti 


I.' 

: 


c 


; i'  r>  _ T;t- 


o.  ■ 


r. 


i . 


> 


. . ''■■  ’'■ 

■ji'  ■ 

/ 

Jr. 

< . - ,;■ 

\>3.i 

18. 


hydrochloric  acid  (r58).  With  either  of  these  methods  the 
maximum  amount  of  amino  nitrogen  is  liberated  from  peptide 
linkage  but  according  to  Van  Slyke  there  is  less  tendency 
towards  deaminization  of  amino  acids,  particularly  cystine 
(39),  at  100°  C.  than  at  150  or  160°  C. 

Complete  hydrolysis  of  5-gram  samoles  of  casein  and  deam- 
inized casein  was  effected  by  autoclaving  for  3 hours  at  124° 

C.  with  200  c.c.  of  3.0  N hydrochloric  acid.  Henriques  and 
Gjaldb8,k  (37)  found  that  under  these  conditions  the  results 
of  hydrolysis  were  anproximately  the  same  as  those  obtained 
by  heating  for  1.5  hours  at  the  higher  temperature.  In  both 
cases  some  undissolved  narticles  remained  after  autoclaving 
and  the  supernatant  liquid  of  the  deaminized  casein  was  color- 
ed a deeper  brown  than  that  obtained  from  casein.  Each  hydrol- 
ysate was  evaporated  to  dryness  on  the  water  bath,  taken  up 
with  water  and  diluted  to  the  mark  in  a 500  c.c.  volumetric 
flask.  Amino  nitrogen  analyses  were  made  on  1 c.c.  portions 
of  the  hydrolysates  by  means  of  the  Van  Slyke  micro  apparatus. 

The  total  amino  nitrogen  of  casein  was  found  to  be  10.08 
per  cent  of  its  total  nitrogen  content  while  9.84  per  cent  of 
the  total  nitrogen  of  deaminized  casein  was  present  in  the 
amino  form.  Since  the  difference  observed  between  these  per 
cents  isprobably  within  the  range  of  experimental  error,  it 
is  believed  that  the  total  amino  nitrogen  of  casein  and  de- 
aminized casein  is  the  same. 

The  Tyrosine  Content  of  Deaminized  Proteins . 


Despite  the  fact  that  some  investigators  have  obtained  a 


■>  'V, 


'.:  i:  i ■:  .'•  OC  •••C'j'.i  :/c.l  .••1 


i.; -v^'  e. 


r,  V'  { •*/!’’ 


' v‘ir-,!'  , 


i »,  « r*  rf. : f* 


I . > i r)  w 


i>4-  fUU^/ V 'X-  i 'i 


■■I  .• 


• .’••■•  / • '.' i , '■’  ' f,  r ■•..■^f T 

■ ii-i  i '''0  •.'••  lSjI  ••£•.'•  .V'.'iJ  r“J  ui.:.  • " 


'lo  .r  ■ 


/ .A 


r •'*  ( 


^ I 

'.  r-<  i 


'yi  H ' t\ e-  :.  i . -1',  t 


r. 


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M ^ • • 

-V,  ' > ♦’ 


'xr'i.r*  j' 


t’,''  ./■■•  ^ .', 


r.'l  :.<;<: ;.-  ‘ i-  i 


j r ;/■.  ,,v,,  ' , t 

'■•  •I 


'w  '*''-p'.'f>Xl  i'.  ^ .^r:Pij', 


;,  .,  f’l;  fK.-5'.-  .:  . 


■!.,"■  Ci  r*' 


■ , C"'! 


li.;  7*  J"  •-,V 


"Of’  r'  n ; :v'i:vT  t»rO'  ’^'f  .•.T 


'f' Off-^jj'vv  . 

’ T'-r  ; c . ^ -.^f.  - i , •'*>'''  cTfe?.  '■’^  S' ' ' < f“ir.s 


' 'i;  0'  r\j^y  vvf:‘'  ' •'  ’M'-  .•;  y;c ''Afnl  .•  .V  C f'**  /?OT 


.1 


OrMtcl  ■ ' r ‘ ' ■ ■’■  f.  ft' 


0 


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p. 


.ri  .i  u ^* *^ ■ *' ' 


’ f.-  ■>; 


it-r.  Ir: 


,■  .1 


■■’Wiir*  ; '.  v' 


• i : 


0<  r>.  : c \ s^.. '•■ 


■ .<> 


u-^stiuoi: 

I 


V > ■ 


..JV.’;*  V*  i '.„ 
iAV«  ill  " 


19. 


positive  Millon’s  reaction  with  deaminized  proteins  the 
statement  has  been  made  that  "Von  den  Monoaminos&uren  ist  zu 
sagen,  dass  ihr  Gehalt  quantitativ  wahrscheinlich  unverS,ndert 
hleibt,  mit  Ausnahme  des  Tyrosins,  das  bei  alien  Desaminido- 
nroteinen  fehlt,"  (26).  This  conclusion  is  based  largely  upon 
the  work  of  Skraun  (l)  who  could  not  obtain  crystalline 
tyrosine  from  deaminized  proteins  although  other  amino  acids 
were  isolated  in  amounts  comnarable  to  those  present  in  the 
original  protein.  Since  deaminized  casein  was  found  to  give 
a positive  Millon's  test  in  the  present  study,  the^resence  of 
tyrosine  would  seem  to  be  indicated.  That  this  assumption 
was  correct  was  definitely  proved  by  the  following  experiment. 
Ten-gram  samples  of  casein  and  deaminized  casein  were  hydrolyz- 
ed for  12  hours  with  200  c.c.  of  20%  sulphuric  acid.  Some  of 
the  humin  which  was  formed  was  removed  from  the  hydrolysates 
by  filtering  on  fluted  filter  papers.  The  sulphuric  acid 
was  neutralized  with  a suspension  of  nure  barium  hydroxide 
and  the  resulting  white  precioitate  of  barium  sulphate  was 
filtered  on  fluted  filter  pacers.  After  concentrating  both 
solutions  upon  the  water  bath  until  a scum  of  barium  salts 
began  to  form  ucon  the  surface  of  the  deaminized  protein 
solution  the  excess  of  barium  was  removed  by  precipitation 
with  dilute  sulnhuric  acid  solution.  Since  a water  clear 
filtrate  could  not  be  obtained  the  barium  sulphate  was  allowed 
to  settle  out  in  tall  cylinders  and  the  clear  supernatant 
liquid,  which  was  tinged  slightly  yellow  in  each  case,  was 
siphoned  off.  Both  filtrates  were  concentrated  upon  the 


, ,,  t"'  ;^.'.-u  ; vJ^j#fjr;.3U  i-  III 

V 'W'  '■■  ’’ 

' .*.  V.c'^'  •*'• 


■ ' ’ , (T  'i..  ' ->i  '( 


V . i-.tjii  1 1111*1^ 


. 


■i  ,'.  , T-l 


,;  bt»5  ■/:r-iitr.f'  f:-  ' <*:bL) •..;•■  -r 


;r.;^  .’ 


r;- 


u ..!  ' /•r.r tif;-  . rri;b  .*y.r\  *'  i.t'r’v*  f n 

*'\'V'  v I.' .{.»r .! ••*  I :-:c^;r  .rit:i0* 

't 


k-.,  r>  »i  r & 
S*' 


;o  ir  twcffo 


!y 


•y*.  ‘'i  ',  ^r; 


- ::r.T  , (Hi^y  X:'oy  fro  i-b--rcr 


‘.'.n’j  cfi'’  jc-'  / • j w-  •?’■  •'?;'■?:  /.-I/ 


’ bP:'i;  •:-  ' . ' vrb  ' .•  ^7 n f ^ •*'  :r  //V I «•; * .*•'.  tW 

?■  / I . ■ _ ' ■ 


I, 

J 1 

r. 


t;  .b  r:;l  w-v-'ccryff#  ■ r.-  .?«J  ' -oSi.hn  r • 

:<■:  r . ■ ' 


-X J rC.  r «'...  ::•■  i 0"^ev.' 


'“i'O 

f.’ 

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if  i '•y7'^.i‘\t'.i  t. 

; .i  cf,,/ 

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.. 

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1* 

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■;ti'<T.'.,;o  ■ ■' 

iw 

w ^.  • \ j 

1. 0..  .. 

b w.-j.'-T,:  :'>^,  .'Of:  i 

L'.’ 

f 

1 

.'.'Li.T'-  ■ jb'. 

z 

i'.bi.yo‘X' 

: : • 'fix  ••  'i  0 r <:  ■;' 

'"fV  vjs 

c’f'Sy.^  n 

■ ' ■ ' 

rr-.i  Z .11  Jf  • ' O.L.r.bV'  .v/j'Xb'Off  e'  .1 

■’  ■ *T  ' - ■ 

. c cj  rx^chy.  'y.uotf',  ..  . 

- i'  ’ 

b ■“>  o':)Ci‘T . '.<■.  bjSvj 


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' . ' ■ |<4 


■ ,m,4  •■  w ' , ^■'11 


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’•a*'  i" 

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f<i  ',  'r 

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F.  1 

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V « 

' ' ' 

•y  ;;,  rfr;..;:-  •/'•Suro  Ci^  . : '•'  riv^i/L':  r-  ^ 

'■'  ■ ' ' ' 

£’0  X£=:  r/jl-t  -:::  ‘‘t  r npi;  ■■:  ^ b; ; r.  'rtb «KJ  . Tn;  .-^|§L 


.n*  wO*ir  b-  :;  X. 


v:.. 

f'ir r M/.'ftvv. 

^ • ■ ' ' m' 

• f.0  . 

Lor,  t'iofi  ■ ' 

iijr  ;.;■■« .-I'-M i.ri-;  ?■■./<;.-  i,V  4.or;  j.>-J 

r •.  ■ ..  • ■ 


: ■ K'^J  ' ' "y;-  ' -'I  ^ i;;':*  - 

\.  ' .!•'  ':  -'■/ 

/ ' .V  rijof.'.f  i. : X;-,  .".tfj-b 

‘ / I*  « ' V<‘ 


Vi 


1 

20. 

water  bath  until  crystallization  had  begun.  After  standing 
overnight  the  mother  liquors  were  filtered  on  a Buchner 
funnel  and  the  crystals  of  tyrosine  sucked  dry.  The  impure 
tyrosine  from  casein  was  tinged  a grayish  white  while  the 
deaminized  product  was  yellow  but  after  recrystallization 
from  hot  water,  the  tyrosine  obtained  from  each  source  was 
pure  white.  No  effort  was  made  to  obtain  a quantitative 
yield  of  tyrosine  from  casein  and  deaminized  casein.  However, 
the  amount  of  this  amino  acid  isolated  from  the  latter  protein 
seemed  only  slightly  less  than  that  obtained  from  casein. 

The  amino  acid  isolated  from  casein  and  deaminized  casein 
was  identified  by  its  crystalline  form.  In  addition  the 
color  reactions  with  Millon's  and  Mftrner's  reagents  were 
positive. 

Prior  to  1912,  the  quantitative  estimation  of  the  ty- 
rosine in  proteins  was  carried  out  by  isolation  of  the  pure 
substance.  Abderhalden  (40)  found  4.5  per  cent  of  tyrosine 
in  casein  and  this  is  the  figure  which  is  usually  quoted. 

In  1912,  the  discovery  that  the  blue  color  given  by  a solu- 
tion of  phospho tungstic  and  phosphomolybdic  acids  with  phenols 
was  specific  for  the  phenol  group,  led  to  the  development  of 
Polin’ s (41)  colorimetric  method  for  the  determination  of 
tyrosine  in  proteins.  Although  tyrosine  is  the  only  amino 
acid  in  the  protein  molecule  which  has  this  groaning,  Abder- 
halden (42)  found  that  this  blue  color  was  also  given  by 

tryptophane,  oxy tryptophane  and  oxy  proline.  However,  Johns 
and  Jones  (43)  have  shown  that  the  blue  color  with  tryptophane 

was  less  intense  than  that  given  by  an  equivalent  amount  of 


k '•*- 


r*i:  -' ' , 

M 


' - . - y r.  '1-- 

t f 

V » * 

, ^ 

■ '/ 

'1..  i 

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k .'  : J A ; 

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'■/:  Oi:'  . 

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■^'  fv: 

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V i ;■ 

/'it'  " '.« tvi  J ■,.  ■■  ' 

1 . ■ , • 

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1 

XC'iOVi-  rj'' 

. r*'!  ' -'i 

■'  < 

pV 

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'"'C  ■'  '.j .'  _N'. . ■ 

J!  jO 

• ^ • V t-'  ' 1 •'  • ** ' 

•/  'i.r  1’’’  a.' 

:vili'’:  e*iL-v,  t;-'t'.''-i'JL J-'i  "©r f i?  ‘.  irnf'ivo  -.1 

^•r^^••^MVv•  •' o r'.  r,-ciTv 

^ .•:■./ to?*’/  t.j  " ,v> 


Jorf 

, y 1 


J:  - ‘^r  ,7 


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:i 


V '■  ' Divine 

■ i- ■ 


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I., 

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'iv 


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; 

, t.  'J.  fv 


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i ' J ' ' ■ ■^.'■ 

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' ' ’ ' ' '■  ' . ^ r,  '.  ,31 


■it. 


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jrv 


¥ 


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I 


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■ ^f  i . " ' ^ 

.'1 

• ' :'• ' ■■  . ' ''•■  • *.'4  c i f-rt.k 


Jt”  ■ I' I j r>n'irx....‘  i ll-l 

• ‘ >/'  ‘i 

*.>'•; 'u  .'•  ■'••  t '■  ■>  c'rr*  ''  i . > ' . 


J,C' . — o: >vV-.>  , ; .p  K^j. ■ .*  .V  ,.  I :f; .;■ ' «/  ' ■'•'•■•  . ••••'  •'  ■ .<■'  t:\‘  Mr>©'''V' 

-ivTc';- i rj  • J5f^ 

c t i '.  V . ^ f ■ '. I '■'•■.■■ : f''=r.  • V n ¥ r>.u‘'3 <f» y^'l , 

Uv',,  ' ' -I  , 


't  . 


21. 


tyrosine  while  decomposition  products  of  tryptophane  gave  no 
color  at  all.  Therefore,  these  authors  concluded  that  re- 
liable results  for  tyrosine  in  proteins  can  be  obtained  with 
Folin's  colorimetric  method.  Gortner  (44)  has  recently 
studied  the  action  of  Polin' s phenol  reagent  upon  cure  amino 
acids.  This  investigator  found  that  tryptophane,  prolin, 
cystin,  histidine  and  valin  gave  the  characteristic  blue 
color.  The  other  amino  acids  which  reacted  with  this  reagent 
were  known  to  be  impure.  As  a result  of  this  study,  the  con- 
clusion was  reached  that  the  colorimetric  estimation  of  tyro- 
sine in  proteins  is  accurate  only  in  the  absence  of  trypto- 
phane and  other  easily  oxidizable  substances. 

The  average  figures  for  the  gravimetric  determination 
of  tyrosine  in  27  different  proteins  was  calculated  by  Folin 
(41)  to  be  2.647  per  cent  while  that  obtained  by  the  colori- 
metric method  was  5.065  per  cent.  According  to  Abderhalden 
the  values  obtained  by  the  colorimetric  method  are  too  high, 
while  Folin  believes  that  the  gravimetric  figures  are  too  low. 

In  all  probability  the  true  figure  lies  somewhere  between 
these  extreme  values. 

In  the  nresent  investigation,  the  tyrosine  content  of 
casein  and  deaminized  casein  was  determined  according  to  the 
colorimetric  method  of  Folin  and  Denis  (41).  The  results  are 
given  in  Table  III. 


22 


TABLE  III. 


Time  of 
Hydrolysis 

Percent 
Casein  1 

of  Tyrosine 
Casein  A-' 

hours 

12 

5.56 

3.83 

16 

5.98 

4.03 

19 

5.78 

3.57 

Average  5.77  3.82 


It  will  "Quoted  that  the  average  figure  for  the  tyrosine 
content  of  casein  was  found  to  be  5.77  per  cent  and  that  this 
is  .73  per  cent  lower  than  the  value,  6.50  per  cent,  found  by 
Folin.  Johns  and  Jones  (43)  believe  that  some  tyrosine  is 
destroyed  if  acid  hydrolysis  is  continued  longer  than  12  hours. 
The  data  obtained  in  the  nresent  study  do  not  support  this 
contention.  It  is  evident,  however,  that  some  of  the  tyro- 
sine or  the  substances  reacting  with  the  Folin  reagent  has 
been  destroyed  during  the  process  of  deamini zation,  since 
the  tyrosine  content  of  deaminized  casein  was  found  to  be 
only  about  66  per  cent  of  that  of  casein. 

D.  The  Distribution  of  Ni trogen  in  Gasein  and  Deaminized 

Casein.  Owing  to  the  inadequacy  of  existing  methods 
for  the  quantitative  isolation  of  the  amino  acids  of  the  oro- 
tein  molecule  coranarative  determinations  of  the  distribution 
of  nitrogen  are  of  considerable  value  in  the  characterization 
of  proteins. 


tl^f  -"H  ‘ 


. . . ^ 

-«Cii.-«L%4j  if^-  ..nncMHiMiM  ‘ 

^ ■■i 


^ r.'  f 


'i.  . ■:l':'^::-.  ' ‘ ,1a' ''yiii 


•s:  :'»T!.v;!^"''''^-,.  m 


, V.- 

t I , 


,'.;  •n' 


<1  nVl 
‘ 

*‘k  ' ! 


►V  ■■ 


'•^sc. 


'j*i «, 


r .■■'* 

4 


L ■ , ,.> .-,.  ■ ,TM  ' I 

I ’ !■'  ^ 

*"■'  ■ , -■■ 

,-  ■^'  ',' iv,!  I 

r « #''*-  -»^  "'■  •-Va*'^'-’  • \V'  "«<iyici^'^  “ ',v  *»  '•' 

,<f  ■^'  »-*  4-i'  ' ‘^'  T 

•^/r^..'.5j^i;.A*ii;-^i.\  s ^'<3.  >ffir  aIS® 

r,vi  ■ ' * 'V  **  ■ -i,  T-  .•  ^ '■■■'•  ’ ■ *■  'm\>  ,»  '.  vriva  lie  ^ 

K v-i  ■ .,.  ', '.  - V'  . •■;■■■'  i ’“'Eji;  V-  ' -■'^'  ' ■: 

70‘Elffc  rV’l  „ . 

bhjjri  "'iV  iifi  i.^''C^s';*:i-  ,.tjrjjj^\t  ^to^X  4'pric"-*tofr 


ii* 


i , ,p  i!?-. .,. , ,,^,i'.v  , a- ■ 


l'  - ' V'-*  '■  * L -,  — ^ ''*  I , *■  ■ k>L  ■ ' «• 

Ult’'' ■ ■■-,■*  V'  ‘ '5^  ’ • ■ - ■'  a « v""-  ' 

^ r elc'*  .u/ip<3fj!x/£.  ’’r  ■'j'f  ’ ffi  • 

' '*'  ' 'i,  ^ { <•  - '/-i  ‘k  'j  *'-'•■•.•,  ' ':4^  &•; 

&'  *’-*^  • v^,(^SfV'  VA fc  vmx^f:  ,S;r ' ki  ^ X*i  - rr(>i-^ ftWroc^iife-  •*- 

r*?..‘  <■  ,'v;''  *'..  '.\^  - - vifti'  •;’•:•'»  ...  ';'4lH&1^f-' 


' i»v.,.i  ‘ .‘  ‘■■T'yi 


1^  ; 


r ' •'.  ••  t ■.*'  . ‘_  • -'^  - & .;  '.-15-, 'v  r "'.k'ft  * . •’;,  .1-^  ■■■■.' 

> pcf  a4  -ffi¥f a^ . 'tKlAi  jgpffi  ?j 


f-.,  1, •*>-‘' . j , , . ' , , • ■’,  j'..,  : ■ .' > ■ 


t » 


EKH 


4 a»a  - jit  .'iiifi'Ss  i^i^taja^.-afc„  -ajirf. 


I 


. sV 
Ju'>0 


vwt 

/I  ' ■ 


^ ^ ^ - ' ,4  . ■'  *-  \y^\ 

' . '■  ',,-  ■ ■ ‘'  • ;'- ' - ' ' "'  ,;  •' 


•jj'i 


jRiCf’jrb'r  ©irL/y^bicr  nlatfj 


'■  ii . ’V,  1'-  'Y  ' ' '„  'V‘ 


2'3. 

In  1899  Hausmann  (45)  determined  the  amide  nitrogen, 
the  di amino  nitrogen  and  the  mono  amino  nitrogen  of  various 
proteins.  Many  criticisms  (46)  of  the  methods  used  in  esti- 
mating the  nitrogen  nresent  in  these  forms  have  "been  made 
and  the  value  of  the  results  has  been  questioned.  The  chief 
difficulties  seemed  to  lie  in  the  accurate  estimation  of  the 
amide  nitrogen  and  in  the  quantitative  separation  of  the 
hexone  bases  and  cystine  from  the  mono  amino  acids.  Imnrove- 
ments  upon  the  original  method  of  Hausmann  and  that  developed 
by  Kossel  and  Kutscher  (47)  culminate  in  the  well  known  Van 
Slyke  (48)  procedure  for  the  partition  of  protein  nitrogen. 

Although  in  general  the  Van  Slyke  procedure  was  followed 
in  the  nresent  investigation  of  the  distribution  of  nitrogen 
in  casein  and  deaminized  casein,  certain  modifications  v/ere 
incorporated  from  unpublished  results  of  Grindley  and  Hamil- 
ton. In  each  case  6 grams  of  casein  or  deaminized  casein 
were  taken  for  dunlicate  analysis  and  these  samplos  were 
hydrolyzed  in  the  usual  manner.  It  was  considered  to  be  ad- 
vantageous to  distill  off  the  excess  hydrochloric  acid  in 
vacuo  before  removing  an  aliquot  portion  for  the  estimation 
of  total  nitrogen.  By  the  use  of  a dual  system  for  vacuum 
distillation  duplicate  determinations  were  ’’ade  under  exactly 
the  same  conditions  of  nressure.  The  apparatus  for  arginine, 
as  modified  by  Grindley  and  Hamilton  permits  the  passage  of 
purified  air  through  it  during  the  entire  six  hours.  Air 

under  pressure  is  passed  through  a purifying  train  of  con- 
centrated sulphuric  acid  and  concentrated  alkali  to  remove 
carbon  dioxide  and  ammonia  and  thence  to  the  bottom  of  the 


;«■:  t ■ • 


i ■ » Zx 


!»'  • '’  '>  !?v 


t,  . V t-  I '*  -i  ■ \ '-'  ■ i»  ■''  . 


i j 


X. 


>' ; 


V,. 


filJDi.^^V  '5v.  ■.(;|  opimjfe  .■<?cja«rr'.tVf<i 


Kite'  i,$L^  cjp^f  - ^ f ’ ' ‘ ‘ 


'■■  "'■''■  . .'  \ \,*  '--''a,  - '5  - ' ■ 


,F  ■ e/k»’  4q  x*^zV^.Pr»,. ‘^'K''  '^'*r .?  f A 

//  A<Jr  . *’v.  ! , ' -vl..  , \-' -'*f  ■ , . . \ 1l  /-'■■* '-^V  .i"  — 

«tt!i^i«6;if!;?!i'  . ?;5icsi>*'  ■ f^nir.Vt  :.Xa/%>fll'-'  • fpCl^  ,tp  s^'E^^dl.  . V 

TRi-'"  ^^' .-■  V ■’  ■'' ' 

v_  . : ','V‘^Ka  , 7’’ TJi*3l.v'^  '■■■’•'’ 


■Mr  - ■/  '"-  ' ' ',  ‘ , ■ S‘  ■ ' ', 

' 'i»^pS'4»  pii,i.f'^^-.sf»  'tfi'  efi;A3::2 ; 'V  v W ■ . 


24. 


Kjeldattl  flasks  by  means  of  a capillary  glass  tube  which  pass- 
es through  the  stonoer  in  the  Kjeldahl  flask.  It  was  found 
that  if  a bubble  of  air  per  second  is  allowed  to  escape 
from  the  Folin  bulbs  that  the  last  traces  of  ammonia  are 
aerated  into  the  standard  acid  and  subsequent  distillation 
as  recommended  in  the  original  nrocedure  is  unnecessary. 

It  has  recently  been  shorn  (49)  that  arginine  loses 
one-half  of  its  nitrogen  as  amino  nitrogen  by  the  action  of 
nitrous  acid  at  20®  C.  for  2 or  3 hours.  This  observation 
leads  Sekine  to  believe  that  the  formula  for  the  calculation 
of  histidine  nitrogen  should  be  3/2  (D-  l/2  arginine  nitrogen) 
instead  of  3/2  (D-3/4  arginine  nitrogen).  Although  the  de- 
tailed renort  of  this  investigation  is  not  available,  it 
would  seem  that  the  action  of  nitrous  acid  upon  arginine  for 
2 or  3 hours  is  not  comnarable  to  the  30  minute  reaction  em- 
ployed in  the  Van  Slyke  procedure.  Therefore  there  appears 
to  be  no  justification  for  altering  the  formula  for  the  cal- 
culation of  histidine  nitrogen.  In  the  present  investigation 
the  Van  Slyke  form.ula  was  used. 

The  figures  for  the  distribution  of  nitrogen  in  casein 
and  deaminized  casein  are  given  in  Tables  IV  and  V.  On  the 
basis  of  these  figures  the  percents  of  arginine,  histidine, 
lysine  and  cystine  present  in  these  proteins  was  calculated. 
These  results  are  given  in  Table  VI.  The  incomplete  recovery 
of  nitrogen  in  the  first  analysis  was  probably  due  to  losses 
in  mono  amino  nitrogen.  ■ The  analysis  for  casein  agrees 
rather  closely  with  the  figures  quoted  in  the  literature  (50) 


1 _ , •*  • ' ?f  :7  " ; ■ i ‘ ■*’  ■'  \ ■ '■■  ' i.  .., 


<l  • - 


’’iNM -"'‘••«d4(io«l 


-y ; v'^' 


'pr  i 

i*.^vvt*>i:v.!*'?-w  ,<:  ..',.i:5^ /'i  ■■Sr/'.^.M. ■{ 

r>.  fT4>.  . Vtf ~ r«.  41;  ..  1 <n#  ' ■ 


rlr>€=^4  it . fer.poit^f;  li. (t,  'll?- , i 

■ ^v  ■:,  '-i^  ' '■ ' ,^-,:/v  ■ ■ V 'iS  ^'.  ■..-«-:%i- 


;•■- «%3' 'iu  i^^.l  -^>19-  ■ g^oi*! 

'V  ' ',  '■'  1^,,'..  ; 'f  :.(!  .,  ' . ' ' **  '■•^  * ",  5‘  ^ • *‘  ” *' 


‘m<t 


vf^S 


W'. 


m ^ 


' ■ ^ '<  «'■  . V''  ' ^ ■'*■  . ' ■'  •■.r  ■iJLfl«t?'.  ' 


,__  _ , , - , ■ - , B-W'^ 

( Sift'  Tvb  iiti’^'J ;#  e!l  '8i’«:?^*'7t;|tvr3|r« 

■ -r^'O;  .-^<5  irr jjiij:,  - i jsi-14  t • *‘r  •^flKfi 

,;...  f...  ,\:  .^r/'  ■ ,:/  ,:,„"-t'-''^  ■ \' 

' ff. ' ^'’ 'tia' I 


4 ,:: '.1/  '•>  ^ '>  a,  ■■  . ..:^^Wi''^/r  ‘ 


;at , 6 j/: 


1^- . td>  'm-V  .. 


.— i.i'TT 


25 


TABLE  IV. 


The  Distribution  of 

Nitrogen  in 

Casein 

No.  1 
Ni trogen 

No.  2 
Nitrogen 

srams 

per  cent 

srams 

per  cent 

Amide 

.0906 

10.26 

.0916 

10.49 

Humin 

.0173 

1.94 

.0190 

2.13 

Arginine 

.0720 

8.15 

.0673 

7.42 

Histidine 

.0533 

6.03 

.0537 

6.01 

Lysine 

.0637 

7.21 

.0812 

9.09 

Cystine 

.0064 

.73 

.0043 

.48 

Mono  amino 

.4225 

47.84 

.5253 

53.78 

Non  amino 

.0788 

8.92 

.0530 

5.93 

Sum 

.8046 

91.08 

.8954 

100.33 

Total  nitrogen 
by  Kjeldahl 

.8830 

100.00 

.8935 

100.00 

The  figures  given  in  determinations  1 and  2 renresent 
in  each  case  duplicate  analyses. 


> i 


___  - , , - . , . K'  Tv 

'^‘•: ' v.V;FiKfawv 


'.•5'  . ■'  - ' icS'  ^f''"'*-  T ■^'^  ' '.'V''  ' A '..  .^- 


IP''' ^ 'W» 


"j.:.„'.i«2rf5ia  itlass.  -. 

(<p  ‘ - . ■'  '‘wrvr. , ' ' .ifv  '•  ■ 

- . - - , ; 

^■-  i’>  .>v  ' -a'  »r'-  ’■■ 

it’ '"  ’,t ' ( ■•  ' •^  ir 

-^'  '.  I 

',*V 


m 


p. 


^J. 


-—  Ji 


g^'A.0^0.- '■■  . ■ ^i^2^«A^Si||! 

" ; 'SV,t^^;s  . , kMiniA^ 

t:ch '# ■ "^'- ■ 


5'.''  . “■  ■'  * ' .4  ■^•''^■■.*  ^o^oox.  f.ali^&.i 

i’ 'v  - .r>-  — t ' 


't  ■> ' ^ ■'  ^ ( j .■  I 

' r 


-'V-  Wj  '•  ' -r 

. .: . ‘'"■’f#  1'.  : 'fj^l 


<t.  <1 


t#  1 


bT  ' * 1 


■y± 


I ."^j 


• •J'/ 


I’W^  ' ■■■,.  y"--  't’ 

Sr-^  '/kA/'  ■/klTJfcifcauja.r-. , ,,  ■■.>->  .-1,  -m  T 

■ 1 itJilfiari  III  I |M|  I g*M  Bii^  iiiti‘' ti  I I'liii  iiiiiir'i  « III  fall!  i I'mi  i i ■■'  -“-  ’ - - . . ..  . 


Lk)iL>  .1 


r'H 


ii‘'  'tf  ' 


26 


TABLE  V . 

The  Distribution  of  Nitrogen 
in  Deaminized  Casein. 45- 

No.  1 No.  2 

Nitrogen  Nitrogen 


grams 

ner  cent 

grams 

ner  cent 

Amide 

.0921 

11.11 

.0921 

11.09 

Hum  in 

.0178 

2.14 

.0257 

2.85 

Arginine 

.0608 

7.55 

.0588 

7.09 

Histidine 

.0554 

6.68 

.0525 

5.39 

Lysine 

-.0054 

-.40 

.0056 

.67 

Cystine 

.0070 

.84 

.0022 

.26 

Mono  amino 

.4295 

51.78 

.5520 

66.50 

Non  amino 

.0859 

10.12 

.0486 

5.85 

Sum 

.7465 

90.02 

.8155 

98.20 

Total  nitrogen 
hy  Kjeldahl 

.8290 

100.00 

.8500 

100.00 

45-  The  figures  given  in  determinations  1 and  2 represent 
in  each  case  duplicate  analyses. 


'■  »F*>T  ■’• 


r*. 'T{1  •’ 


vf<  r 


’"f 

•1;.*.'^  -■ 


V-, 


iwi  ■ f 


I ' ■ , '■ 

'V  /•  ; ^:-  ^ ' .J  '"  ■' 


M'-l  ■)■ ->'^> 


C' 


,'  . » 


' \ • 
,■.  \'> 


</ . 


MX 


'*  1#!  '!45\  PfiO  i'TJU’ IC?  y 

xK'cflLurfi'O  (iJt  , ’• 

. i" -O’'  ' " .'.'L  ■l(&‘s. 

••  ■'W^*-'*'  y§L__'  : , -Jk  ■ ■ 


» ■■ 


s''*  ' ''  ’ '/ V ’’■^  , 

? ‘ L ■■■■'^3^'-’^  rcR'c.  r ■x'-^'w-"'' ■ ’ «ro.  ,'■  ■ •#^rul:wi!'i'%'' 

..-'  W-  - - , ■ ,;•  ■ - . . " ,:  ('-  .-MV'.,, 


•'’'  ■■  «0:.'7. 

*»■... 

M”’,.''  ' ■ , .V: 

‘"■'l*.  H U'  ■ 


f*BdO. 


* ' fl!7*  P 


i 


3 . 


T^. 


^c*r- 


z'' 


'V 


' r 


■rr.'i'ir 


, RSes.  ^ .' 

*»  Oi^^laA^rron. 

'■'cu.ap'<  . ;■ 

rfr»Tir:*,4i  I ' , . ' ' , ' 'it|^f'  •<  / r Hi' 

?*  ■> . ' ••  „ ■:-  ■ •;<  -^^Sl  '!<3BK?;'  S 


■ »?sSv  >-■ 

Ik  ' ’ " ■‘y‘i»V  ’^' 


, vr  ,T  -■ 

v>.  *t»i 


>■ 


K:*  *' 


aO‘X>ar> 

^-_  .:.m  =1  , -.,....  p ■ * . ' ^ . a ' -.  ^ y . ^ 


■ ■ > --f . .■  .V :.;  ’ .. . 


I'?  • 

K'W 


p.  ’VJ 


£.('  ' i' 

'''Pl 

■V',tS-’  ■ '.S'. 


K.  Ki-y^ 


' <i> 

’ ir-  •••••  Mti3ni  ' '’W^  ■ ' 


,■  < 


v:  i'.ii  . 


'•' 'A  , : .r  . 


,9  ' i aV--  ' -.f'_ 


IVi'^r’^-.r^T  ; K-  ,■ 

i.U 


■ '■  ; ^ 


■ - -F^  ■ - - •^- I.  ■ 

' jiirt- iHii'  '^v?!  ■ ■1..'^  .^^  ^.-rA4^2'SJ 


f I 


27 


TABLE  VI 


Deainini  zed 


Amino 

Casein 

1. 

Casein  A-64 

Acid 

1 

2 

Ave . 

1 

O 

Ave 

Arginine 

3.72 

3.44 

3.58 

3.14 

3.03 

3.08 

Histidine 

3.27 

3 . 30 

3.28 

3.40 

1.98 

2.69 

Lysine 

5.53 

7.05 

6.29 

-.29 

.48 

.09 

Cystine 

.85 

.57 

.71 

.93 

.28 

.60 

- I I"*'  i\i^^|iKpir<i  itfi-i^i-tritf 

r f».  n ,^^.m  „ '•  ■ 

<•  : ia?:,  . ‘ ‘^■'  • ■.'(■f^ 


\ , . v'''''"^v"'  ; 

w'  >■  .-i'- • ■*  ■ ’ 

I ..;  .■4..'.^,v**' ..  nc  •*,.  «»*#•''• 


'■^  • 


: ."lip,  . 


■ ■r  ‘'^-'^r;i 

i'fi  ¥ \ '■'*/*’  ■< 




iVi 


irri. ;. 


A<  . 


-*vSH!3i.olut'9i_i  -' ' 

i.. , . . ,i-''  •■*  ■ iisiiiifflK*-' 

, *■' , -1^'  ; l^' 

_ "■  •.  . ~ . . ' ' ',.  * . .‘^'  ■ * ■ 1 ''}■■  ^ ' ’'it?  • ' V .:’ 


I ■■  *:if.- 


•■’1  •»! 


CO.  ■ ■ ■ B^'. 


. ^ "S' 

,'■'.{  I • ■"'.1  ' . ^.  '■  ■ I ■ '.Btk-  ^ I ! I 


, , 'T 


: ! ’O.  a;?.'  y^  Ofl  - t™. 

«W,*.  / -LJ  • - SB*’ 


•"'-  (inW«iiC 


’p'-' -•  smrmm 

F-  • ^ :■ 

«.!'  : Ibt'L' .f>V'';&  ■■,  - ' . .>;iSp|ltJ 


•-<.  M"  W >4/  -i.!.  . 


•Ii-Ji 


. A -V'-i  .M.,1  • . ‘ r . ,’•'  , ‘!;.£fisifp  • ..„*5^;:->Wtl 

■'=i.,'v4  fr>V'  -I 

? j-v-'i^rv', 


' '.1.  •'  .'"■■■  •■  i'l.- '■  , -'^<7.*  V:,V-  '•  ' 'iSWKtr  ' 's*’i^'3 


n v^^JT 


^•‘.  ' './;  ■'■  V** 


' ■ ■ Pi; . '!iV; 


i/^'4  ?'■'  f f 


1 


• P*:-  :*w -’’..a^S'  i^VJ.  'CW !»■.•. '^'...V  /■•■f.'  'HU  >-•'  '., 

1"^'  ^ Jvp-4|jk|  1 


;-.v'Y 4 


L-:i43»3*T(.»"' 


28. 


and  in  general  the  figures  obtained  for  casein  and  deaminized 
casein  agree  closely.  Skraup  has  renorted  a diminution  in 
the  per  cent  of  arginine  and  histidine  nitrogen  in  deaminized 
casein  and  the  entire  absence  of  lysine  in  the  deaminized 
product.  In  the  present  research  the  average  figure  for  two 
determinations  of  arginine  nitrogen  in  deaminized  casein  v/as 
found  to  be  7.21  per  cent.  Since  this  value  is  only  .56  per 
cent  lower  than  the  average  figure,  7.78  ner  cent,  for  casein 
it  is  believed  that  this  difference  is  within  excerimen tal 
error  and  does  not  signify  a destruction  of  this  amino  acid 
in  the  deaminized  product.  This  is  what  would  be  exnected  if 
nitrous  acid  is  without  action  upon  the  guanidine  group  of 
arginine  which  has  been  shovm  to  be  free  in  the  protein 
m.olecule.  If,  on  the  other  hand,  it  is  true  as  reported 
recently  by  Sekine  (49)  that  the  guanidine  group  of  arginine 
is  slowly  attacked  within  the  space  of  a few  hours  by  nitrous 
acid  at  low  temperatures  some  destruction  of  arginine  would 
be  expected.  In  the  first  determination  of  the  histidine 
nitrogen  of  deaminized  casein,  6.68  per  cent  was  found,  while 
by  the  second  analysis  only  3.89  per  cent  was  obtained. 

Since  the  former  value  agrees  approximately  with  the  average 
figure,  6.02  per  cent,  found  for  casein  while  the  latter  is 
only  about  one-half  of  that  amount,  no  definite  conclusion 
concerning  the  fate  of  histidine  in  the  deaminized  product 
can  be  drawn.  The  average  figure  for  lysine  in  casein  was 
found  to  be  8.15  per  cent  wrhile  in  the  deaminized  product 
minus  .40  per  cent  v/as  obtained  by  the  first  analysis  and 

--  ■ — 


29. 


1 

I 


plus  .67  per  cent  "by  the  second.  The  average  figures  for 
these  determinations  is  plus  .13  per  cent.  It  is  considered 
that  probably  no  lysine  is  present  since  the  amount  found  is 
within  the  limits  of  accuracy  of  the  method.  It  would  seem, 
therefore,  that  this  observation  is  in  harmony  with  the  work 
of  Skraup  (1),  in  which  no  lysine  could  be  isolated  from  the 
hydrolyzed  products  of  deaminized  proteins  and  with  the  cur- 
rent theory  concerning  the  nature  of  the  free  amino  groups  in 
the  nrotein  molecule.  According  to  this  theory  the  epsilon 
amino  groun  of  lysine  is  free  in  the  nrotein  molecule  and  it 
is  this  group  which  is  attacked  when  deamini zation  occurs. 

It  is  further  believed  by  Van  Slyke  and  others  that 

the  free  amino  nitrogen  of  native  proteins  is  due  almost 
entirely  to  the  epsilon  amino  group  of  lysine  since  the  free 
am.ino  nitrogen  content  of  these  proteins  is  approximately 
one-half  of  the  nitrogen  of  the  lysine  which  they  have  been 
shown  to  contain.  Those  who  oppose  (6  and  7)  this  theory 
have  found  that  certain  native  proteins  have  a free  amino 
nitrogen  content  which  is  not  one-half  of  the  nitrogen  of 
the  lysine  present.  However  the  belief  that  the  free  amino 
nitrogen  of  native  proteins  is  due  at  least  in  part  to  the 
epsilon  amino  group  of  lysine  renders  likely  the  assumption 
that  deaminization  of  native  proteins  cleaves  the  terminal 
amino  group  of  lysine  to  form  oc  amino  e hydroxy  caproic 
acid.  Since  this  derivative  of  lysine  would  not  be  basic, 
its  subsequent  reactions  would  be  those  common  to  the  mono 
amino  acids,  i.e.,  it  would  not  be  precipitated  by  phospho- 
tungstic  acid  but  would  appear  in  the  mono  amino  filtrate. 


'••jrics'r^..*^-..*  .-v’jc~vV'J' ».;."7i 


, h I. 

-.r..'  - . w 


I 


‘.'T* 


^4 


\ 't 


t v'  -V,  - 


,vno- ' f ,i  \ ■..■  'c. 


,'.  n ' I ■;  \'  ' ' r ' ,./  ■■''  v' r ' 'i 

•..  ■ ' . ••’  ■■  ’ . ■ t.  •:  •'  * .V  .,ti 

r 

■ '•■  f * ■■•'.■.  >*"'■''  I i‘r> ■'•.]  yi'c^t  t j ■% 

■•••■  " i,  '•  ^ . .iwi'V 


, ■'t' 


'j.r'j 


• / » .*  • -.  i ' y a 

•'  V 


n j. 


'*  ■ '.  V.  ^ .*  ,j;  ,0  . f 


^ -.'f  oe  ''rj.o'ibw'’’ 

.M  JM’O'  ''uifJ  .■."^>*t 

f 

: ■ ' - c 0-TCr 

>.:xl  r. 


■ '>:  - r 


\ ' 

' 1.W  ' / • 

, , J . i { ; 1. 


*. : : ,1 


' ' ' r ' 

( . 

.'n.  . 


'•lirOfV'  . ^'■!J  i?;. 


s ■ 


J c 'x.'i  . I ;' 

-.r--  i',..  .- f . i"  'r-4  -LO^ 

j < -V  f . ^ ^ 

nowc^  ' ' v.^  . ••■,.; 


' '■t  ' , 


ri/:r£^ 

q-  i:V‘v':.r;  ■>'.■  o,%4  t/'x  ' i*H.i 

' .1 


j'i  ''>.  • ■ 1 


■ ' - 4'»‘  n*"  -i"  ■ ' : '-■■  fi  w t ■'  'J*!  i 3 if ! Cr 


i ”>■•  'j.  <"  ■-',  r'C 3 iT."***'  /■,■■  * 


■'V  vcr;  *■ 


rt 


. <f . '•  1 


) 


r»:’0 


.'I  '':  n' 


'.'■'J'  ( “^  lifu*  *o‘'‘fry;0‘  fc.  ffr/orla 


’.  ' ^ '^,0 

i""  •-■  ■ / •■  w *:o 


'I  r.  ( r 


..,  ,■''.•./■■  pti  3c:-'  - .-  tutsh 

V..;  V », 


.V  ■i'.^  •-  ’•  a ’o,-.  , ‘-UJ  J*  '• 


^r-V.vX 


’ J ■’  :i-  -.,:  •;: 

r,  i '^:.  ' : 1 3 “ J,  .'.  V •' 

c*‘-  •> 

. : - . .rr'  ■•.  ■■  , 

' ■’  ' ,v  ’■('  f*'  1-C.’  ' ,, 

- 'r^a7-jO*^f:  ■.•, ‘qji:  ifr 


» 


.a  jv ; 'f  c ; r > 


■'  3.^n'3 


.!.’'•  •• . •■’•i''7  I' J.  :v^X  ^ qijox',-- 


:jt:  »i;;  • I"- ., -u'l'r:  i. 


■ n ■ ' ' 

J 


-'•/i 


'“'fj-''  V POf^r  rx  :'-X:uv.v  3;.'/. 


3 ' ' 


loMiiii 


; j.*  j 


»r„ 


9'X 


31/ 


30. 

The  marked  increase  in  mono  amino  nitrogen  in  the  filtrate 
hy  the  second  analysis  indicates  that  this  or  some  other  ly- 
sine derivative  has  become  associated  with  the  mono  amino 
acid  fraction.  Attempts  made  by  Skraup  and  others  to  isolate 
this  hydroxy  derivative  of  lysine  have  failed.  Skraup  (22) 
claims  to  have  obtained  the  anhydride  of  oc  amino  S 
hydroxy  valerianic  acid  which  he  believes  has  been  formed 
from  the  hydroxy  caproic  acid  derivative  of  lysine. 

In  the  nresent  investigation  it  seemed  likely  from  di- 
rect determinations  of  tyrosine  in  the  original  acid  hydroly- 
sates (see  Table  III)  that  some  of  this  amino  acid  had  been 
destroyed  in  the  process  of  deaminization.  To  obtain  further 
evidence  bearing  unon  this  point  analyses  of  tyrosine  in  the 
mono  amino  acid  filtrates  of  casein  and  deaminized  casein 
were  made.  In  the  case  of  casein,  '5.41  and  5.89  per  cent 
were  found  while  2.7  2 and  2.98  per  cent  of  tyrosine  7<jas  found 
in  the  mono  amino  acid  filtrate  of  deam.inized  casein.  It  is 
evident  therefore,  that  a considerable  part,  about  50  per 
cent  of  the  tyrosine  or  the  substance  which  gives  a blue 
color  with  the  phenol  reagent  has  been  destroyed  during  the 
process  of  deaminization. 


u-r.T»,' . *.  ‘I  » ;.jr-  iia^ 


l-^T’ 


k'^3^^1  ''**'^  ^' ' ' *'t' . 


HT5 


? '‘  V ',*,'»!  ■;  ,=  -i  \, 

1, 


W M;  ;7 j;-' ■ V vfT 

&F‘‘***'**^^  i?W  jr,t‘':rte&>t4fr.;'piUirit  Qm>{r  Y*!  ! s>jJ»gj»*rl»j  ‘a*j}“iS,-effC^ 


w '4  '■  ■ }■''''■  ’•  ■ ' -.j  ‘ '■'‘'-I,'  ^ ' 'f  ' 

:;^‘-r:“tr^ori-^0.  '.te  *ra  (?inj  .?.orw-  .»-e ?.u dnoo^-.  :i 

. I •,  ,'i  ■ •'■lY  ..  -:■  ' ’.■'  - ^ , '.-w  •'  ■•'*  . 


.’  'Vj'  i^.:  ^ ■>  '*  '■  I .j*  ' ' »i‘f'^3, 

;,  • ■ sr.tl  ^(f  in  ! 

::  ;:  • ■•  : . . / “4. ,.  ^ ' ,..  •• ‘-i:- . ki.  ' '■ 

nii'  * . - . ^ 


0®*''y*93b^<|,  OAito-  6-Xif{'j  ?Q  ■■'  ‘•-'- 


|^'l''.tiJ  , :i  ''  . ■ . 4; 

* y <1^  4*1^;; ' r i O^"',- 


^ r • 


Sv'  '*X'0  JT^'30  '(S'*' 

i>'f(.  ,.  ■ ; ■'  ' ■ ■ '•  , . . 

‘rftl\it^  , (^i-ei^ii09^yi^.in  ^-3 

• ..  , • _ i '*  - » 

;.'•/  . •!•' v-r' 


M 


' <<•.■  .•' >v  / j^.*'^  ' - ‘-jSbr  • ' 

.■’."®‘',Z''a  fv,-e.  ..^ih,-:,>  ^6  Sc”'. 

<v>  ?«5..  feiXliri^' 

cmcm 

y i'«4.<l,  OS'  Jjro<fa  ,;yW-  ia-'f 

iif,['-  ■ r"  ,'Vy..  *--A  E 

jt-  * ’ . < i..  -’  ‘ I*'  V-  - . ...^  ^ 

' *’iI^.Y^A'=  V'*  ' ' "^  , '''C-  '.,,  u 7^"'  \ .:'  ^ '"  ' t'  - J 

f ■,  .:,  ,■ ' ...  W--^  * 

'?Yv ’ ."..  ' .(  ’*i,v«  X '.  (•.•->'  '•iYjfVw''''  ‘m’ 

^ '/  -..I'aa.  *<  ^.S  Jt  MT-. -*C.  .i  i 'tirmJlaB’  . 


Si*t  ‘ .' 

, vr'if^M.yjjlSS 

l^^.u  •.  .♦  ,;r  ..f-'’-’  ^ li^ikltfeAis  . I •...  .i.  / 'fi.  /jk^i 


31. 


— 

V.  Digestion  in  Vitro. 

The  observations  renorted  in  the  literature  in  regard 
to  the  digestibility  of  deaminized  proteins  are  conflicting. 
Treves  and  Saloraone  fl8)  reported  that  deaminized  proteins 
were  not  digested  by  artificial  gastric  or  pancreatic  juices, 
while  Schiff  (16)  found  them  to  be  completely  digested  by 
dog's  gastric  juice  although  the  rate  of  digestion  was  much 
slower  than  with  the  original  proteins.  Levites  (26)  found 
digestion  to  be  complete  with  dog's  gastric  juice  except  for 
a small  residue. 

In  a review  of  the  methods  used  for  studying  digestion 
in  vitro  the  statement  is  made  by  Prankel  (51)  that  "the 
best  index  of  the  extent  to  which  a protein  has  been  disin- 
tegrated is  the  ratio  of  the  amino  nitrogen  at  a given  time 
to  the  total  amino  nitrogen  obtained  after  hydrolysis".  Of 
the  various  methods  by  which  the  amino  nitrogen  of  nroteins 
may  be  estimated  Prankel  chose  the  Van  Slyke  procedure  as 
"best  suited  to  th^problem  in  hand."  After  examining  eight 
methods  for  the  study  of  proteolytic  action,  Sherman  and  Neun 

(52)  concluded  that  "the  quantitative  determination  of  

the  amino  nitrogen  of  digestion  products  appears  to  be 

more  delicate  as  a means  of  detecting  proteolysis  than  either 
the  biuret  or  the  ninhydrin  reaction  and  more  delicate,  ac- 
curate, and  generally  applicable  as  a means  for  its  measure- 
ment than  any  of  the  other  quantitative  methods  here  studied." 
These  authors  used  the  Van  Slyke  method  for  estimating  amino 


•:  . ; Vi  - 


.j  i-, 


ilZ) 


V,;j  'o£;; 


“■  ..u  n'c.t'f.: 


m 


; 'I*  Iv  nl 


c'*'  - ‘io  ' *■?  'Vo 


K/: 


: o.S  '6r<j  Vj 


■ Y.f  ;jq1}i'^v  (tiiJ 

■ .t  i .1*  •-• 


, VC,  ^ s;{^IV  ,fVt  ' o e'f 

• -.  ‘’.Of:/!-  'dJ^  i -.iift 

' ’ '■  ■ ' ' \ ' • ' L'^/  ' " ■ ■'^.T'  . ' r 

hLD/,,[."^i$  r-  ^ "^•.  . T T'-' . i ’ 0 . r ^ »v  •.'j'Jjj  • ■ V-':  fibc/.'J  *ir 

^'  r '••■•^  ’•V'r'f-'fo  srJ ''  ' Vz-r'i  te-r/.r-  '--cv  '{OiL) 

o3  l:  I*  *_r'i  '-?  .-.j’  vLv.i'^cr  ' j ;:Aau;ji ‘j  ;VtO>  i.''i  o:.’’:n>'-  orfj 

Vo  -i.:v 

^ -■ .. .f  ■ •■' 'V-'’  ■ ^ ‘ • :•  ■ - 

■ tT’.  r.s.'  i^'Tor  rip.  J'-.  o Or<r,,>/fNr{  r . ■ ; ,.-j 

y 'o i 0 o c i f rr<7T?  7*" 


• .‘c>  1 0;;  J ,=?•  £'^  j ff  3 lyo  ■ j v n*' 


, vrJ.yjl/0 

:M  } . 


V ■ ^.•^.£Vrf6Up  ' ;or'  Vr  ’.  - r \t.Te«’ 


^ .'t  ij, nc  bCiVo  •j''f 


ii'f'V-  0^':iJj  'V  -/i5 


I 

32. 

nitrogen  but  assumed  that  results  with  the  S^irenson  method 
would  run  parallel . 

In  the  proteolytic  studies  of  the  present  investigation, 
the  action  o^epsin,  trypsin  and  erepsin  alone  and  in  series 
was  studied.  The  samples  of  pepsin  and  trypsin  used  in  these 
experiments  viere  commercial  preparations  known  to  be  active, 
while  erepsin  was  nrepared  from  the  intestinal  mucosa  of  a 
dog  according  to  Prankel's  (51)  modification  of  the  method 
originally  outlined  by  Rice  (53).  The  erepsin  preparation 
was  considered  to  be  trypsin  free  since  it  was  without  action 
upon  fibrin.  The  samples  of  casein  and  deaminized  casein 
were  hydrolyzed  according  to  the  method  of  Henriques  and 
Gjaldb8.k  (37)  but  the  total  amino  nitrogen  was  determined 
by  the  nitrous  acid  method.  This  figure,  A^'/hen  corrected 
for  free  amino  nitrogen,  was  considered  to  represent  the 
amino  nitrogen  in  nentide  linkage,  i.e.,  the  maximum  amount 
of  amino  nitrogen  actually  available  for  liberation  by  enzymes. 
The  liberation  of  amino  nitrogen  during  digestion  was  follow- 
ed by  means  of  the  Van  Slyke  micro  apparatus.  Prankel  ran 
controls  ”with  all  reagents  and  ferments  in  the  same  quantity 
except  that  no  protein  was  added,”  to  correct  for  the  amino 
nitrogen  present  in  the  reagents.  In  the  present  study  con- 
trol experiments  were  carried  out  with  the  same  amounts  of 
protein  and  reagents  but  using  boiled  enzyme  solutions. 

With  the  latter  tecnique  corrections  are  made  not  only  for 
the  amino  nitrogen  present  in  the  reagents  and  enzyme  added 
but  for  the  free  amino  nitrogen  content  of  the  proteins  and 


i - -i!. . - ;..  

;i27' 


B»;\’  Co  fJu£re*T:  vr'^W;lC24 


* *•; 


.33. 


for  that  which  may  have  been  liberated  by  the  hydrolytic 
action  of  the  reagents.  It  is  believed,  therefore,  that  the 
corrected  values  for  the  free  amino  nitrogen  are  an  accurate 
measure  of  the  amino  groups  actually  liberated  by  the  digest- 
ive action  of  the  enzymes  employed. 

The  results  given  in  Table  VH  were  obtained  by  the  si- 
multaneous digestion  of  5.0  gram  samples  of  casein  1 and  de- 
aminized casein  A-64.  These  proteins  were  suspended  uniformly 
in  250  c.c.  of  .2f^  hydrochloric  acid  and  20  c.c.  of  an  aqueous 
solution,  containing  .2  of  a gram  of  pepsin,  added.  Controls 
containing  the  protein,  all  reagents  and  boiled  enzyme  solu- 
tion were  run.  After  thoroughly  mixing  the  contents  of  each 
flask  5 c.c.  of  toluene  were  added  as  a preservative  and  in- 
cubation at  38°  C.  begun.  At  the  end  of  3 hours  incubation 
the  casein  sample  had  gone  entirely  into  solution,  but  the 
deaminized  casein  and  the  controls  had  settled  out.  The 
supernatant  liquid  of  the  controls  was  colorless  but  that  of 
the  deaminized  casein  sample  ?;as  colored  yellow,  indicating 
that  a partial  digestion  had  taken  place  in  the  latter  case. 

At  the  expiration  of  the  3 hour  period,  uniform  samples  from 
each  flask  were  taken  for  analysis  of  amino  nitrogen  in  the 
Van  Slyke  micro  apparatus.  To  5 c.c.  portions  from  each 
flask  was  added  .5  c.c.  of  N sodium  hydroxide  to  stop  the 
digestion.  The  resulting  solution  was  diluted  to  10  c.c.  in 
a volumetric  flask  and  2 c.c.  taljen  for  analysis.  At  stated 
intervals  in  the  digestion  of  these  proteins  subsequent  amino 
nitrogen  determinations  were  made.  At  the  end  of  110  hours 


• 7 T♦*^4V,_  '•■. 


■ V-v.V.3C^^.  ■.'.^.  • '.’^V''  W-'  ••  'iv  • • 

w-  '/  n«:.^^r^.' sii'V!^' ’”«4> 

|A  ' ' '*  *'  ' <*  J _ , ■ j,  . '^■^''',tY,-f  *'■  j^.'’'‘4*> '" 

H' • < ’ ' ■ ■ ‘'■>*wi>  . L K'o  V, X (fCi;  r 1^ v A'-ilij, . 


':r, 

" ' «.  1 1 ^ I- 


’ V i^j! 


s;  k >v- 

•'•1.-'  ■ ' ...  •,.' . ^ _ 'i,.  ■ ' . '' 


f4  ■ ' 

- i.  ■ ‘ ^»|- 


,;*■ 


S'  ,^^‘V 


»v<  V , ,\ 


rf,  ■ » 


r f - ■ A.  . '*-»i'  * V tf  ''’*■  'S 

•V  ! '-rv:  ^ ^ '«i.  r ■ ".  v.^-" 

-r—  i ,j  ^ <L*»  rf  *.  _4  xV<r.*  A'  . . ' . « • ._  ^ "w  • i -:  V •'  -i_j  . . . uV  - «.  flf  -u 


i 


34. 


TABLE  VII. 

The  Peptic,  Tryptic  and  Ereptic  Digestion  of 
Casein  1,  and  Deaminized  Casein  A-64. 

The  Percent  of  Total 
Amino  Nitrogen  Liberated. 


Enzyme 

Hours 

Casein  1. 

Casein  A 

Pepsin 

0.0 

0.0 

0.0 

tf 

14.5 

9.6 

2.6 

ft 

38.5 

11.1 

3.3 

tf 

86.5 

11.7 

3.3 

tf 

110.5 

11.8 

3.3 

Trypsin 

12.0 

53.2 

25.8 

tt 

36.0 

68.3 

30.9 

tf 

60.0 

79.9 

33.4 

ft 

132.0 

78.7 

33.5 

Erepsin 

17.5 

91.7 

56.2 

tf 

41.5 

92.1 

65.4 

ft 

65.5 

95.8 

65.8 

ft 

89.5 

95.7 

65.4 

160  200  240  2B0  320  ^0 


The  Peroent  of  Available  Amino  Nitrogen 


rt  h 


.4 


t r 


36. 


digestion  200  c.c.  aliquots  from  each  peptic  digest  were 
taken,  20  c.c.  of  15^  sodium  carbonate  solution  containing 
.4  of  a gram  of  trypsin  added  and  incubation  at  38*^  C.  begun. 
Boiled  trypsin  solution  was  added  to  the  controls.  After 
several  hours  the  deaminized  casein  sample  went  into  solu- 
tion giving  a brovm  but  transparent  liquid  but  there  appeared 
to  be  no  change  in  the  controls.  At  the  end  of  132  hours 
digestion  with  trypsin  150  c.c.  aliquot  portions  were  removed 
from  each  flask,  20  c.c.  of  erepsin  solution  added  and  incu- 
bation continued  for  89.5  hours.  Boiled  solutions  of  the 
enzyme  were  added  to  the  controls.  For  the  determination  of 
amino  nitrogen  in  the  tryptic  and  ereptic  solutions  5 c.c. 
aliquot  portions  were  used.  After  arresting  digestion  by 
the  addition  of  .5  c.c.  of  glacial  acetic  acid  these  samples 
were  diluted  to  10  c.c.  in  a volumetric  flask  and  2 c.c. 
used  for  the  analysis  of  amino  nitrogen. 

Similar  experiments  were  carried  out  with  trypsin  and 
with  erepsin  to  determine  whether  these  enzymes  could  attack 
deaminized  casein  without  the  preliminary  action  of  pepsin. 

2 gram  samples  of  casein  1 and  deaminized  casein  A-64  were 
dissolved  in  120  c.c.  of  sodium  carbonate  solution. 

Casein  1 gave  a solution  of  medium  opalescence  while  casein 
A-64  form.ed  a deep  red  solution  in  which  gelatinous  particles 
were  suspended.  20  c.c.  of  a solution  containing  .2  of  a 
gram  of  trypsin  or  erepsin  dissolved  Inja.  ,5%  sodium  carbonate 
solution  were  added  to  each  flask.  10  c.c.  of  an  alcoholic 
solution  of  thymol  were  added  in  each  case  as  a preservative. 


> , - . ; V’TTTffl?  ^ w 

T’  "S'-  ,,-'''’W'r 


. »?»*.!''#.  . ■ »■  T Akiii-\  .-  >1.^:.  ^ L _i'.  i.  ^«;  -i ' o -I  ^-Tifi  . 'i  . r .'iji-  ' ■*  i»‘ 


.£Ti 


l » , y= 

I'  '-'j «*•«.’  ‘^,r*o b'i.fKr^_ io^}vUii  Wt ^ ,^1 

t |T 

■*':  *.  .LTC  ■ : ; ^ _ * M • '■  5 ’ . * JD  ^ ^ J 

i .:.iv4i<o-f‘  f)vOfi,ti%^  nrtf'i  ' 

■"  'i  ' ■*  ,-  - .'  * . -'-w',  V 1 '>  /.'  » <•■  '».ii.^*  n. 


• ‘ " ' ^‘  ' 

* • ^ ' t,Ji^  * > 


^ 1 ;/ ’ iJTi ii.-  V, ,'■  \iS 


ii.S.  ....  ...  _.. 


37. 


After  incubating  at  38°  C.  for  22.5  hours  a 5 c.c.  sample 
from  each  flask  was  taken,  .5  c.c.  of  glacial  acetic  acid 
to  arrest  digestion  and  the  resulting  mixture  diluted  to  10 
c.c.  in  a volumetric  flask.*  A clear  but  brown  liquid  in  the 
case  of  casein  A-64  indicated  some  digestion  had  taken  place 
in  each  instance.  The  appearance  of  the  proteins  in  the  con- 
trol flasks  was  not  altered. 

The  results  obtained  from  the  enzymatic  hydrolyses  of 
casein  agreed  closely  with  the  observations  of  Frankel  (51). 
Pepsin  liberated  11  per  cent  of  the  total  amino  nitrogen  of 
casein  in  87  hours:  trynsin  superimposed  upon  the  pepsin  di- 
gest set  free  79  per  cent  in  60  hours,  and  by  the  further 
action  of  erepsin  95  per  cent  of  the  total  amino  nitrogen  was 
liberated  in  66  hours.  These  values  are  maximum  for  these 
enzymes  since  the  amino  nitrogen  was  found  to  remain  constant 
over  a period  of  25  or  more  hours  of  additional  digestion. 
Frankel  believes  this  to  be  due  to  the  auto  destruction  of 
the  ferment  rather  than  to  the  inhibiting  action  of  the  end 
products  as  suggested  bjr  Abderhalden  and  Gigon  (54).  The 
digestion  of  deaminized  casein  proceeded  in  every  case  at  a 
slower  rate  than  that  of  casein  and  the  total  cleavage  was 
considerably  less.  Only  3 per  cent  of  the  total  amino  nitro- 
gen of  deaminized  casein  was  liberated  after  110  hours  of 
peptic  digestion  in  contrast  to  11  per  cent  with  casein. 
Tryptic  digestion  for  132  hours  set  free  only  33  per  cent  of 
the  total  amino  nitrogen  as  compared  with  73  per  cent  for 
casein,  while  the  further  action  of  erepsin  liberated  only 


U.‘  ' ■' 


• : I. 


itV  ,•  » • f 

n V ' I o ' 

tiO.-  J.  , f 


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65  per  cent  of  the  total  value  for  amino  nitrogen  as  con- 
trasted with  95  per  cent  for  casein. 

It  is  possible  that  this  difference  in  digestive  action 
hetv/een  casein  and  deaminized  casein  may  be  due  to  the  less- 
ened solubility  of  the  latter  and  its  consequent  less  in- 
timate contact  with  the  enzymes.  There  is  also  the  possibility 
that  reactions  incidental  to  the  process  of  deaminization 
may  have  taken  nlace  to  alter  the  peptide  linkage  in  such  a 
way  that  its  cleavage  by  enzymes  became  more  difficult. 

It  is  evident  from  the  results  reoorted  in  Table  VIII 
that  trypsin  will  digest  deaminized  casein  without  the  pre- 
liminary action  of  pepsin.  However,  the  rate  of  digestion 
with  trypsin  is  slower  than  that  with  the  preliminary  action 
of  pepsin  and  with  trypsin  alone  a lessened  cleavage  is  pro- 
duced. The  digestion  of  deaminized  casein  with  trypsin  is 
less  extensive  and  proceeds  at  a slower  rate  than  that  of 
casein . 

Since  it  is  known  that  casein  is  attacked  by  erepsin 
without  the  preliminary  action  of  other  enzymes  it  was  of  in- 
terest to  test  the  action  of  this  enzyme  towards  deaminized 
casein.  It  was  found  (see  Table  ’ IX.)  that  11  per  cent  of  the 
total  amino  nitrogen  of  casein  was  liberated  after  93  hours  of 
ereptic  digestion  but  no  amino  nitrogen  was  obtained  from  de- 
aminized casein.  It  would  appear,  therefore,  that  erepsin 
does  not  attack  deaminized  casein  directly  or  at  least  it 
does  so  very  slowly. 


;,<t  -'‘Tf  A.,-' 


*<'5*Wr,P' 


^'\‘^'iV:.»..>  .. 


•;',  -r^?3  •.'..  ■y:,^.-i:)isi..  .-/-Jito  %.l’-  ofti/jy'  .Mtey 

mxvis  ' ■<  s.  . ' ■ 'I’v?*'  ■-':  . --v\  -i 


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■ft 


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j*  (‘f  '*'  ' * ' \ ' * ' ' ' ~'^"  " ' "' '' 

\s:  . Jo  ( ^^”5  -j^iX^^'Vtft^'is.)'  V^Sv. 

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Bfei  ‘%i.‘  • ;. . j*:'  .r'r. 

■ viiM '■;-*5|^^i^fe.... __. .. ... 

' " 'P  •'-'  »J( ^ |iS»^ Tiru‘ r (MiiTYiitffiii|-iina  y j r » 1 1 ii'.y  priityjiiji  limimnaiiny  i 


39 


TABLE  VIII. 

The  TryDtic  Digestion  of  Casein  1. 
and  Deaminized  Casein  A-64. 


The  Percent  of  Total 
Amino  Nitrogen  Liberated 


Hours 

Casein  1. 

Casein  A.-64 

o 

# 

o 

0.0 

0.0 

22.5 

41.3 

25.3 

45.0 

50.4 

27.9 

68.5 

52.7 

27.3 

92.5 

52.4 

28  .7 

TABLE  IX. 

The  Ereptic  Digestion  of 
and  Deaminized  Casein 

Casein  1. 
A-64. 

Hours 

The  Percent  of  Total 
Amino  Nitrogen  Liberated 
Casein  1.  Casein  A-64 

o 

• 

o 

o 

• 

o 

0.0 

21.5 

00 

• 

o 

o 

• 

o 

45.5 

00 

• 

o 

0.0 

93.5 

11.6 

o 

. 

o 

II  >i<|f|M>ii»iii'tt  i»i,  .5i  . 'X..  .wi*.,. 


I j ^ *ii  ' * ' I'  ■ • : t ^ ^ i 'i_  „.  /''’/'•■  h-'  V 

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/'V  '.-'■« 


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Ty.  V ^ V rfftkp:  :!  •'<  i ^ ■ , ■ kA|i4y| 

• ;’.>vi  •■•  ‘i  ■’  • ajJL’  '.v^ 


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r ■'O'  ^'• 

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} 1^ 


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B .rV'K  . ..'i.  v.^u  B., 


I'b 


I • \: 


40. 


Bn 

F.  The  Behavior  of  Deaminized  Casein  in  the  Animal  0rg;anism . 

Since  in  the  nresent  investigation  digestion  experiments 
in  vitro  indicated  that  deaminized  casein  was  digested  al- 
though at  a slower  rate  than  casein  it  was  desirable  to  study 
the  behavior  of  deaminized  casein  in  the  animal  organism.  A 
female  dog,  weighing  about  9 kilos  was  maintained  upon  a uni- 
form diet  for  10  days  to  permit  a constant  level  of  nitrogen 
excretion  to  be  reached.  At  the  expiration  of  this  period 
10  grams  of  deaminized  casein  A-64  was  added  to  the  standard 
diet  and  the  elimination  of  extra  nitrogen  in  the  urine  de- 
termined. 

The  standard  diet  as  given  in  Table  X was  considered 
to  be  calorifically  adequate  for  a dog  of  the  size  used. 

The  beef  heart,  chosen  in  preference  to  other  kinds  of  meat 
because  of  its  uniform  composition,  was  dissected  free  from 
fat,  valves  and  tendons  and  ground  to  a pulp  in  a grinder. 

The  mass  was  kneaded  to  a uniform  consistency  and  preserved 
in  glass  jars  at  a low  temperature.  Since  in  feeding  experi- 
ments requiring  the  use  of  a synthetic  diet  it  is  important 
to  render  the  food  as  palatable  as  possible,  the  standard 
diet  used  in  the  present  research  was  prepared  as  follows: 


Substance 

Table  X. 
Amount  (fi-ms) 

Calories 

Bone  ash 

10 

Sucrose 

35 

140 

Starch 

25 

100 

Beef  heart 

50 

120 

Fat  (lard) 

25 

225 

Water 

400  c . c . 

— 

Sum 


585 


f t 


1 


OJ 


. .1 V '■  •■ji’  ' 


*r’.' 


s 


I 


■ \ 


■\' 


■ " .,■>.■ 
• • - 


','1 


r w.  ' . C 

iv>  i ^ rn^^lv 'ni 


- ;^V  ’''  ■■.■;  ■ ; '■:«»•.•.:  J:  u\rc  ri^uoffi  ' 

*■  Z'  "■  ■ •' '■  • • ^ .^'*' -' ■'  r<'^  (.  ■■  H • i 


41 


Date 

Weight 
of  Dos 

TABLE  XI. 

Total (1)  Total 

Sulnhur  Nitrosen 

Urea  and 
Ammonia  N. 

% of  total  N 
as  urea  N. 

kg. 

gms . 

gms . 

gms . 

Jan.  7 

— 

— 

" 8 

— 

— 

— 

— 

— 

" 9 

— 

— 

— 

-- 

— 

” 10 

9.90 

— 

— 

— 

— 

" 11 

9.90 

1.355 

.841 

62 

" 12 

9.88 

.068 

1.342 

.910 

67 

" 13 

9.88 

.069 

1.251 

.757 

60 

ft  14 

9.86 

.070 

1.427 

.872 

61 

9.82 

.077 

1.955 

1.622 

77 

" 16 

9.75 

.064 

1.363 

.946 

69 

” 17 

9.75 

.082 

1.246 

.960 

77 

" 18 

9.75 

.073 

1.299 

.968 

74 

" 19 

9.73 

•35-  10  grams  of  deaminized  casein  A-64  were  fed  in  addition 

to  the  standard  diet. 

(l).  Thanks  are  due  Miss  Lucie  E.  Root  for  the  sulphur  de- 
terminations reported  in  Table  XI. 


’fMT.v  ■■  , y ■'  y 'Wiry  s 


i;  :■  !>■  ■.(.,; •O'-  v*>;-t,  ‘’;if  va 


'■'.  -vsi , v -i-.i':/  ■ JJ 

k 'i-'si..'  > , '■  iijj.1  4' 

ulia  I , S ■,  . \ »lL'AI**.’.'  1 


> -I^ifffastl,.  ■ 4'u5iS3,tiit,  • giiS.  . ft  5ii*s 

■“'  » ' '•’  i»l  ■•,  . 1 ••'}  I ^*s»BUi.  '/,■' 

'„X‘  ■ 


.,  ■"  ,v:t  4'\- 

-,'  'ivT.>-'."- 


tL' 


■y  '- 


~“v 


'■'  ■ ■•'  V ".f^^ 


L-- 

P- 

<*I  'T  '• 

blj 

':  w.’ 

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■ ^ ‘C ",  s-(5.P* 

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■ '0 


■|)  ’^,‘  •'<'  '.^'  '■’  ' 


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.'  j,;  - -ss-i.  f^i-r 

iy.  -'ii  i 


A- 


• X‘-  , 


\ * * . 1 ‘ • r.  • ‘ . ■ fi  •»  r . '»  : . 


.#  " '. ..  ' ■■  ' • 


r ; -t 

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' <'  'i'fer'T'.  ' •‘JtOCo  r 

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I aK/'*'.  ' ' -u-  ‘ . V-.  * ■"♦  'i. 

> ' ' ' „•'“'  , '•  . 

ft .1  ,1  ' w .*.'<•■.■  I-!  ■ ■ :'■  '.  I ■ 

t_  ,^.  '. .'  ;.'  • • ' ''5d ■ , '.'  ^ f'.S “t  HBtf>'v', 


- A-'  ■'  '"W' ■ '^f  ■* 


3^:^' 


JJ  i. 


if  . „ , ■ 47  . , ‘ Im/,5iM‘.,V  .)  , i.'V'  ' iMjaJra  ) ^ “ i';-  V-  , - 

''■■  1 . .'  r'  «' ' 'i-  y"  • ,«v  ■ i J '<,4*f5lilM  f I *, 0 ‘M/’  .'5t'' ■>  Si''.'  /■• 

■#.  .,  , *./iv  v.i.'i  '•■  ■ •'  ..  if'"  I •'  V • • -.*|.  ’.<•; . );-*r  ■"’i  , ■ iiftn  'v’>u  ■ 

r-"- •'  1 - IM  Iii1r>ini0««i*4ai11it.  .lalfci  I '^'iLr  I..  I “ . .1 


42. 


10  grams  of  bone  ash  and  35  grams  of  sucrose  were  added  to 
350  c.c.  of  distilled  water  and  the  mixture  heated  to  boiling. 

To  this  mixture  was  added  25  grams  of  starch  made  into  a 
paste  with  50  c.c.  of  distilled  v/ater  with  constant  stirring 
to  prevent  burning  of  the  starch.  In  a few  minutes  the  mix- 
ture thickened  because  of  the  swelling  of  the  starch  granules. 
While  the  vessel  containing  this  mixture  was  cooling,  50  grams 
of  beef  heart  and  25  grams  of  lard  were  added  and  stirring 
continued  until  a uniform  mixture  was  obtained.  As  a routine 
procedure  this  diet  was  fed  immediately  after  catheteriza- 
tion of  the  dog  at  9:00  A.M. 

According  to  the  figures  given  in  Table  XI,  the  total 
nitrogen  elimination  for  the  experimental  day  after  feeding 
10  grams  of  deaminized  casein  represented  43  per  cent  of  the 
nitrogen  added  to  the  standard  diet  as  deaminized  casein. 

This  amount  was  .615  grams  in  excess  of  the  average  nitrogen 
elimination  for  the  fore  and  after  periods.  Since  there 
was  a corresnonding  or  even  greater  increase  in  urea  nitrogen 
the  assumntion  that  utilization  of  the  deaminized  casein  in 
the  animal  organism  has  occurred  seems  well  founded. 

j 

Since  the  preliminary  experiments  seemed  to  indicate  ! 

i 

utilization  of  deaminized  casein  in  the  animal  organism  it  i 

was  desirable  to  determine  whether  an  animal  could  be  main- 
tained in  nitrogenous  equilibrium  upon  deaminized  casein. 

The  dog  used  in  the  preliminary  experiment  was  put  upon  a 
standard  diet  the  nitrogen  content  of  which  was  determined  | 
by  Kjeldahl  analysis.  This  diet  given  in  Table  XII  ^as 


f 


4 V 

’ -wnr^'i  r..i  iiipiii 

BX  i'''-  ” ^1.  ^ a ^ 

v;/- . , 


fcv  . ‘'^T^ 


L'  . ‘ , *■  V . • ’ 


rtf. 


jA  »r  ' L*  - >?/■ 


,,  ' ' ' I < 4'^.' ' . V ■ i ■ ' vl''  -’ ■ ■■‘!-'¥.'"'^  *■  ■''**  ' ■ '•  ■’  ■ ' •%:*' 

f.;^  ■ ■ JM.V.ia-.'  *'  i.-.iL  f >._.  . ,?t*  -,,nt'  . Vi  ■'  fAif 


43. 


comparable  to  that 

used  in  the 

foregoing  exoeriment  although 

it  was  made  higher 

in  calorific 

value  because 

the  animal  had 

lost  weight  in  the 

first  experiment. 

TABLE  XII 

Substance 

Amount 

Calories 

Ni trogen 

gms . 

gms . 

Water 

400  c.c. 

— 

— 

Bone  Ash 

10 

— 

.0002 

Cane  sugar 

40 

160 

.0160 

Beef  heart 

25 

60 

.7400 

Com  starch 

25 

100 

.0180 

Lard 

35 

305 

.0035 

Casein  II 

25 

or 

100 

3.3900 

Casein  A-64 

24.19 

Sum 

725 

4.1677 

After  8 days  on  the  standard  diet,  using  the  routine 
procedure  of  the  preliminary  experiment,  deaminized  casein 
was  substituted  for  casein.  Ordinarily  the  food  was  eagerly 
and  completely  devoured  but  on  the  second  day  with  deaminized 
casein  the  dog  ate  with  reluctance  and  after  2 or  3 hours  time 
the  food  which  had  been  eaten  was  vomited  up.  This  obser- 
vation seems  to  indicate  that  deaminized  casein  becomes 
toxic  to  the  animal  organism.  The  deaminized  casein  fed  on 
the  first  day  was  apparently  not  toxic  or  at  least  not 


44. 


sufficiently  so  to  prevent  its  absorption  and  the  elimination 
of  its  nitrogen  in  the  urine.  No  explanation  for  the  ab- 
normally high  elimination  of  nitrogen  on  this  experimental 
day  has  been  found. 

The  original  intention  was  to  continue  the  experiment 
with  deaminized  casein  for  a longer  time  but  the  toxicity  of 
this  product  rendered  further  experimentation  inadvisable, 
if  not  im.possible.  It  would  seem,  however,  from  this  isolat- 
ed experiment  that  deaminized  casein  cannot  replace  casein 
in  the  diet  because  of  its  toxicity  but  whether  the  deaminized 
product  will  maintain  an  animal  in  nitrogenous  equilibrium 
is  still  to  be  determined. 


• •- ' y '<■  1 ■;.>  ® V- ®T*W 


p 

i^-  ^ ''  ’ .ff  ^ 


*:;■■  -f  T-VS ’ .ct . ' ■ - '•■  ■' 

I - ' '.  ^.  £ •’•'jT  *1  ..  • ito  AJ  '"  , «T  ^ JriiacJ 


'^'-  Lll 


'-yi 


'•j  "’’fv-r' 


f yy  ' ■'. ^ 

■ A'  ^.AS^  M I i-  r . 


□ I*'  rt' 


■^-  J 

■ • " I J V,.*J  -A 

■’  r . 'V,  * '5,'j4^-; 


&’liSj 


■/•■«<  •ihiC'.v  w.’ 

'i\' ' I'i 


Jw''  . A" 

^ 


45 


TABLE  XIII 


Date 

Weight  of  Dog 

Nitrogen  of  food 

Nitrogen  in 

kg. 

gms. 

gras . 

Jan. 

24 

9.75 

4.4177 

M 

25 

9.71 

4.1677 

— 

tf 

26 

9.73 

If 

2.005 

»f 

27 

9.77 

It 

2.240 

If 

28 

9.86 

ff 

2.490 

If 

29 

9.84 

If 

2.640 

ff 

30 

9.86 

If 

2.660 

ff 

31 

9.88 

It 

2.939 

Feb . 

1 

9.88 

It 

3.868 

If 

2 

9.86 

ff 

■}{■  Deaminized  casein  A-68  was  substituted  for  casein  in 
the  diet. 


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m 


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46. 


SUMMARY . 

1.  The  free  amino  nitrogen  of  a number  of  sam.ples  of 
casein,  prepared  in  various  ways,  has  been  determined  and 
found  to  be  constant. 

2.  Deaminized  casein  has  been  orepared  by  the  action 
of  nitrous  acid  unon  casein. 

3.  The  distribution  of  nitrogen  in  casein  and  deaminized 
casein  has  been  determined  by  the  Van  Slyke  partition  method. 
In  harmony  with  the  current  theory,  as  to  the  nature  of  the 
free  amino  groups  of.  the  protein  molecule,  deaminized  casein 
was  found  to  contain  no  lysine.  No  other  notable  differences 
between  casein  and  deaminized  casein  were  found. 

A. 

4.  Tyrosine  was  found  to  be  partially  destroyed  by  the 
deaminization  of  casein  but  not  completely  so  as  maintained 
by  Skraup. 

5.  Casein  and  deaminized  casein  were  found  to  be  di- 
gested in  Vitro  by  nensin  and  trypsin.  Erepsin  was  found  to 
digest  casein  readily  but  to  attack  deaminized  casein  only 
after  the  preliminary  action  of  pepsin  or  trypsin.  In  every 
case  the  digestion  of  deaminized  casein  proceeded  at  a slower 
rate  than  the  digestion  o:^casein. 

The  nitrogen  of  deaminized  casein,  administered  per  os, 
was  excreted  as  urea.  Deaminized  casein,  administered  per 
os,  appeared  to  be  somewhat  toxic  to  the  organism. 


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47. 


BIBLIOGRAPHY. 


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48 


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• 

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Eckstein,  H. 

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49 


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Folin,  0.,  and  Denis,  W. , ibid,  1912,  XII,  239  and  245; 
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ACKN0\7LEDGMENT 


I wish  to  acknowledge  my  indebt- 
edness to  Dr.  H.  B.  Lewis,  at  whose 
suggestion  this  research  was  undertaken, 
for  his  interest  and  helpful  advice. 


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VITA. 


Educational  Career  - 

A.  B.  Simpson  College  1916. 

M.  S.  University  of  Illinois  1918. 

Assistant  in  Chemistry,  University  of  Illinois, 
1917-1918;  1919-1920. 

Fellow  in  Chemistry,  University  of  Illinois, 
1920-1921. 

Publications  - 

Studies  in  Uric  Acid  Metabolism.  2.  Proteins 

and  Amino  Acids  as  Factors  in  the  Stimula- 
tion of  Endogenous  Uric  Acid  Metabolism. 
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